Antagonistic anti-tumor necrosis factor receptor superfamily polypeptides

ABSTRACT

Described are antagonistic TNFR2 polypeptides, such as antibodies and antigen-binding fragments thereof, and the use of these polypeptides to inhibit the proliferation of regulatory T cells (T-regs) and/or myeloid-derived suppressor cells (MDSCs), to expand T effector cell populations or function, and to reduce the proliferation of, or directly kill, tumor cells, such as tumor cells that express TNFR2 antigen. The polypeptides, such as antibodies and antigen-binding fragments thereof, are TNFR2 antagonists, such as dominant TNFR2 antagonists. The polypeptides can be used to suppress the T-reg- or MDSC-mediated deactivation of tumor reactive T lymphocytes, expand populations of tumor-reactive cytotoxic T cells, and/or to directly kill TNFR2+ tumor cells. The antagonistic TNFR2 polypeptides described herein can be used to treat a wide variety of cancers and infectious diseases.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML copy, created on Apr. 28, 2023, isnamed 00786-583003_SL.xml and is 463,710 bytes in size.

BACKGROUND OF THE INVENTION

The use of naturally-occurring and genetically engineered T lymphocytesis a prominent paradigm for ameliorating various human pathologies. Forinstance, while traditional therapeutic platforms for the treatment ofcancer include surgical removal of tumor mass, radiation therapy, andadministration of chemotherapeutics (Shewach, Chem. Rev., 109:2859-2861,2009), the last decade has witnessed a resurgence in the application ofadoptive immunotherapy to cancer treatment regimens. With the advent ofchimeric antigen receptor (CAR-T) therapy, new methods have emerged forthe infusion of autologous and allogeneic tumor-reactive T cells topatients (June, J. Clin. Invest., 117:1466-1476, 2007). CAR-T therapiesharness the resources of the adaptive immune response in order topromote cancer cell cytotoxicity and eradicate tumor material. A commonmotif in adoptive immunotherapy is the use of T cells that exhibit theability to selectively potentiate cytotoxicity in cells that displaydistinct tumor antigens. Examples of this technique include theadministration of tumor-infiltrating lymphocytes (Dudley et al., J.Immunother., 26:332-342, 2003), as well as autologous or allogeneic Tcells that have been genetically re-engineered so as to exhibitreactivity with a tumor-specific antigen (Yee et al., PNAS.,99:16168-16173, 2002).

Despite the promise of T lymphocyte-based cancer immunotherapy, thedevelopment of this therapeutic platform has been hindered by thenatural propensity of the immune system to suppress immune attacksmounted on self cells. Cancer cells express class I majorhistocompatibility complex (MHC) proteins that distinguish these cellsfrom foreign cells. In order to prevent cell fratricide, regulatory Tcells (T-reg cells) have evolved that suppress the activity of T cellsthat exhibit reactivity against “self” MHC antigens. T-reg cellsrepresent a heterogeneous class of T cells that can be distinguishedbased on their unique surface protein presentation. The mostwell-understood populations of T-reg cells include CD4+, CD25+, FoxP3+T-reg cells and CD17+ T-reg cells. The precise mechanisms by which thesecells suppress autoreactive T cells is the subject of ongoinginvestigations, though it has been shown that certain classes of T-regcells inhibit production of the proliferation-inducing cytokine IL-2 intarget T cells and may additionally sequester IL-2 from autoreactivecells by virtue of the affinity of CD25 (a subdomain of the IL-2receptor) for IL-2 (Josefowicz et al., Ann. Rev. Immun., 30:531-564,2012).

Although T-reg cells play an important role in maintaining peripheraltolerance, the same biochemical features that underlie the ability ofthese cells to modulate autoreactive T cell activity also serve toundermine adoptive immunotherapy and the natural immune response bysuppressing the activity of tumor-reactive T lymphocytes. Thedevelopment of chemical modulators of T-reg cell activity has been thesubject of many pharmacological investigations, as access to an agentcapable of inhibiting T-reg-mediated T cell suppression could vastlyimprove the scope and efficacy of adoptive cancer immunotherapy, as wellas improve the ability of the immune system to eradicate pathogenicorganisms that give rise to infectious diseases.

There is a need for improved therapies for treating cell proliferationdisorders, such as cancer, and a wide array of infectious diseases.

SUMMARY OF THE INVENTION

Described herein are antagonistic tumor necrosis factor receptorsuperfamily polypeptides, such as single-chain polypeptides, antibodies,antigen-binding fragments thereof, and constructs. For instance,featured are antagonistic tumor necrosis factor receptor 2(TNFR2)-binding polypeptides, such as single-chain polypeptides,antibodies, antigen-binding fragments thereof, and constructs. HumanTNFR2 contains four cysteine-rich domains (CRDs): CRD1 (amino acidresidues 48-76 of SEQ ID NO: 7), CRD2 (amino acid residues 78-120 of SEQID NO: 7), CRD3 (amino acid residues 121-162 of SEQ ID NO: 7), and CRD4(amino acid residues 162-202 of SEQ ID NO: 7). Antagonistic TNFR2polypeptides described herein include those that bind one or moreepitopes within CRD3 of TNFR2 and/or one or more epitopes within CRD4 ofTNFR2, such as those that bind TNFR2 exclusively within one or moreepitopes of CRD3 and/or one or more epitopes of CRD4 without bindingTNFR2 within CRD1 and/or CRD2.

The antagonistic TNFR2 polypeptides described herein include IgG2isotype antibodies and antigen-binding fragments thereof thatspecifically bind TNFR2 at one or more of the epitopes detailed above.The present disclosure in based, in part, on the surprising discoverythat antibodies and antigen-binding fragments thereof exhibit markedlysuperior TNFR2 antagonist properties when these molecules are in theform of an IgG2 isotype relative to other antibody isotypes. Theantagonistic TNFR2 polypeptides described herein also include those withat least two TNFR2 binding sites (e.g., antigen-binding sites, in whichTNFR2 is the “antigen”), in which the binding sites are spatiallyseparated from one another by about 133 Å or more, as it has presentlybeen discovered that such polypeptides exhibit unexpectedly superiorTNFR2 antagonist effects relative to polypeptides that specifically bindTNFR2 at one or more of the epitopes described above, but that containTNFR2-binding sites (e.g., antigen-binding sites) separated from oneanother by fewer than about 133 Å, such as IgG1 antibodies andantigen-binding fragments thereof that contain antigen-binding sitesseparated from one another by about 117 Å and IgG3 antibodies andantigen-binding fragments thereof that contain antigen-binding sitesseparated from one another by 125 Å.

Also featured are anti-TNFR2 polypeptides that adopt a singledisulfide-bonded isoform and pharmaceutical compositions containing thesame. For example, pharmaceutical compositions of the disclosure includethose containing an antagonist TNFR2-binding polypeptide in which, e.g.,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%,99.9%, 99.99%, or more, of the polypeptide in the pharmaceuticalcomposition is present in a single disulfide-bonded isoform.Antagonistic TNFR2-binding polypeptides that adopt a human IgG2-Aisoform exhibit substantially superior TNFR2 antagonist effects relativeto TNFR2-binding polypeptides that adopt other human IgG2 isoforms, suchas the IgG2-B, IgG2-A/B₁, and IgG2-A/B₂. Thus, TNFR2 polypeptides thatadopt a single disulfide-bonded isoform can be prepared aspharmaceutical compositions and administered in methods of treatmentdescribed herein to promote robust TNFR2 antagonistic effects.

Antagonistic TNFR2 polypeptides of the present disclosure exhibit one ormore beneficial biological properties, such as the ability to inhibitthe proliferation of, and/or to promote the death of, regulator T cells(T-reg cells) and/or myeloid-derived suppressor cells (MDSCs).Antagonistic TNFR2 polypeptides can be used to inhibit the proliferationof, and/or promote the death of, TNFR2- and oncogene-expressing cancercells. Additionally, or alternatively, antagonistic TNFR2 polypeptidescan be administered to promote the reciprocal expansion of T effectorcells, such as cytotoxic CD8+ T cells. This may occur, for instance, bythe attenuation of T-reg cell proliferation and activity or by thedirect expansion of T effector cells, such as cytotoxic CD8+ T cells.Therefore, the designation of TNFR2 polypeptides as antagonists refersto their capacity to attenuate the proliferation and activity of T-regcells, MDSCs, and/or TNFR2-expressing cancer cells and, for clarity,does not indicate antagonism of the T effector cell response. Thepolypeptides (e.g., single-chain polypeptides, antibodies,antigen-binding fragments thereof, and constructs thereof) describedherein can be used for the treatment of a variety of pathologies,including cancers and infectious diseases.

In one aspect, the disclosure features polypeptides, such assingle-chain polypeptides, antibodies, antigen-binding fragmentsthereof, and constructs thereof, that specifically bind human tumornecrosis factor receptor 2 (TNFR2) at an epitope within cysteine-richdomain (CRD) 3 (CRD3) and/or CRD4 and that do not specifically bindTNFR2 at an epitope defined by one or more amino acids within CRD1, inwhich the polypeptide:

-   -   (a) contains a human IgG2 hinge region that lacks a cysteine        residue at positions 232 and/or 233 of the amino acid sequence        of the IgG2 hinge region; and/or    -   (b) contains antigen-binding sites separated from one another by        a distance of at least about 133 Å.

Exemplary antagonistic TNFR2 polypeptides of the disclosure (e.g.,antibodies and antigen-binding fragments thereof) that exhibit theforegoing characteristics are described in Table 1 below. Table 1provides a description of various antagonistic TNFR2 antibodies andantigen-binding fragments thereof as defined by their heavy chain andlight chain amino acid sequences. Antagonistic TNFR2 antibodies andantigen-binding fragments thereof of the disclosure include those havinga heavy chain and/or light chain as shown as Table 1, as well asantibodies and antigen-binding fragments thereof that contain a heavychain and/or light chain having at least 85% sequence identity (e.g., atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.9%, or 100% sequence identity) to a heavy chain and/orlight chain shown in Table 1. Complementarity-determining regions areshown in bold.

TABLE 1 Exemplary Antagonistic TNFR2 antibodies of the disclosureAntibody Light Chain Amino Acid Heavy Chain Amino Acid No. SequenceSequence  1 MVSSAQFLGLLLLCFQGTR MGWTLVFLFLLSVTAGVHSQVQLVQSGAECDIQMTQSPSSLSASVGDR VKKPGASVKVSCKASGYTFTDYLMHWVRQ VTVTCQASQNINKYIAWYQAPGQGLEWIGWVDPEYGSTDYAEKFKK QKPGKAPKLLIHYTSTLESGWVTVTRDTSISTAYMELSRLTSDDTAVYYC VPSRFSGSGSGTDFTLTISSARDDGSYSPFDYWGQGTMVTVSSASTKG LQAEDVATYYCLQYVNLITFPSVFPLAPCSRSTSESTAALGCLVKDYFPE GGGTKVEIKRTVAAPSVFIFPVTVSWNSGALTSGVHTFPAVLQSSGLYSL PPSDEQLKSGTASVVCLLNSSVVTVPSSNFGTQTYTCNVDHKPSNTKV NFYPREAKVQWKVDNALQSDKTVERKSSVECPPCPAPPVAGPSVFLFPP GNSQESVTEQDSKDSTYSLKPKDTLMISRTPEVTCVVVDVSHEDPEVQF SSTLTLSKADYEKHKVYACENWYVDGVEVHNAKTKPREEQFNSTFRVVS VTHQGLSSPVTKSFNRGECVLTVVHQDWLNGKEYKCKVSNKGLPAPIE (SEQ ID NO: 297)KTISKTKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 302) 2 MVSSAQFLGLLLLCFQGTR MGWTLVFLFLLSVTAGVHSEVQLVQSGAE CDIQMTQSPSSLSASVGDRVKKPGASVKVSCKASGYTFTDYLMHWVRQ VTVTCQASQNINKYIAWYQAPGQGLEWMGWVDPEYGSTDYAEKFK QKPGKAPKLLIHYTSTLESGKRVTMTRDTSTSTFYMELSSLRSDDT VPSRFSGSGSGTDFTLTISSAVYFCARDDGSYSPFDYWGQGTLVTVSSA LQAEDVATYYCLQYVNLITFSTKGPSVFPLAPCSRSTSESTAALGCLVKD GGGTKVEIRTVAAPSVFIFYFPEPVTVSWNSGALTSGVHTFPAVLQSS PPSDEQLKSGTASVVCLLNGLYSLSSVVTVPSSNFGTQTYTCNVDHKPS NFYPREAKVQWKVDNALQSNTKVDKTVERKSSVECPPCPAPPVAGPSV GNSQESVTEQDSKDSTYSLFLFPPKPKDTLMISRTPEVTCVVVDVSHED SSTLTLSKADYEKHKVYACEPEVQFNWYVDGVEVHNAKTKPREEQFNST VTHQGLSSPVTKSFNRGECFRVVSVLTVVHQDWLNGKEYKCKVSNKGL (SEQ ID NO: 297)PAPIEKTISKTKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG K(SEQ ID NO: 303)  3 MVSSAQFLGLLLLCFQGTR MGWTLVFLFLLSVTAGVHSEVQLVESGAEVCDIQMTQSPSSLSASVGDR KKPGASVKVSCKASGYTFTDYLMHWVRQA VTVTCQASQNINKYIAWYQPGQGLEWMGWVDPEYGSTDYAEKFKK QKPGKAPKLLIHYTSTLESGRVTMTRDTSISTAYMELNRLTSDDTAVYFC VPSRFSGSGSGTDFTLTISSARDDGSYSPFDYWGQGTLVTVSSASTKGP LQAEDVATYYCLQYVNLITFSVFPLAPCSRSTSESTAALGCLVKDYFPEP GGGTKVEIKRTVAAPSVFIFVTVSWNSGALTSGVHTFPAVLQSSGLYSLS PPSDEQLKSGTASVVCLLNSVVTVPSSNFGTQTYTCNVDHKPSNTKVD NFYPREAKVQWKVDNALQSKTVERKSSVECPPCPAPPVAGPSVFLFPP GNSQESVTEQDSKDSTYSLKPKDTLMISRTPEVTCVVVDVSHEDPEVQF SSTLTLSKADYEKHKVYACENWYVDGVEVHNAKTKPREEQFNSTFRVVS VTHQGLSSPVTKSFNRGECVLTVVHQDWLNGKEYKCKVSNKGLPAPIEK (SEQ ID NO: 297)TISKTKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 304) 4 MVSSAQFLGLLLLCFQGTR MGWTLVFLFLLSVTAGVHSQVQLVQSGTE CDIQMTQSPSSLSASVGDRVTKPGASVKVSCKASGYTFTDYLMHWVRQ VTVTCQASQNINKYIAWYQAPGQGLEWLGWVDPEYGSTDYAEKFKKR QKPGKAPKLLIHYTSTLESGVTMTRDTSTNTVYMELTSLRSEDTAIYYCA VPSRFSGSGSGTDFTLTISSRDDGSYSPFDYWGQGTLVTVSSASTKGPS LQAEDVATYYCLQYVNLITFVFPLAPCSRSTSESTAALGCLVKDYFPEPV GGGTKVEIKRTVAAPSVFIFTVSWNSGALTSGVHTFPAVLQSSGLYSLSS PPSDEQLKSGTASVVCLLNVVTVPSSNFGTQTYTCNVDHKPSNTKVDK NFYPREAKVQWKVDNALQSTVERKSSVECPPCPAPPVAGPSVFLFPPKP GNSQESVTEQDSKDSTYSLKDTLMISRTPEVTCVVVDVSHEDPEVQFN SSTLTLSKADYEKHKVYACEWYVDGVEVHNAKTKPREEQFNSTFRVVSV VTHQGLSSPVTKSFNRGECLTVVHQDWLNGKEYKCKVSNKGLPAPIEKT (SEQ ID NO: 297)ISKTKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 305) 5 MVSSAQFLGLLLLCFQGTR MGWTLVFLFLLSVTAGVHSEVQLVQSGAE CDIQMTQSPSSLSASVGDRVKKPGATVKISCKVSGYTFTDYLMHWVQQ VTVTCQASQNINKYIAWYQAPGKGLEWMGWVDPEYGSTDYAEKFKKR QKPGKAPKLLIHYTSTLESGVTITADTSTDTAYMELSSLRSEDTAVYYCA VPSRFSGSGSGTDFTLTISSRDDGSYSPFDYWGQGVMVTVSSASTKGP LQAEDVATYYCLQYVNLITFSVFPLAPCSRSTSESTAALGCLVKDYFPEP GGGTKVEIKRTVAAPSVFIFVTVSWNSGALTSGVHTFPAVLQSSGLYSLS PPSDEQLKSGTASVVCLLNSVVTVPSSNFGTQTYTCNVDHKPSNTKVD NFYPREAKVQWKVDNALQSKTVERKSSVECPPCPAPPVAGPSVFLFPP GNSQESVTEQDSKDSTYSLKPKDTLMISRTPEVTCVVVDVSHEDPEVQF SSTLTLSKADYEKHKVYACENWYVDGVEVHNAKTKPREEQFNSTFRVVS VTHQGLSSPVTKSFNRGECVLTVVHQDWLNGKEYKCKVSNKGLPAPIEK (SEQ ID NO: 297)TISKTKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 306) 6 MVSSAQFLGLLLLCFQGTR MGWTLVFLFLLSVTAGVHSQVQLVQSGAE CDIQMTQSPSSLSASVGDRVKKPGASVKVSCKASGYTFTDYLMHWVRQ VTITCQASQNINKYIAWYQQAPGQGLEWIGWVDPEYGSTDYAEKFKK KPGKAPKLLLYYTSTLESGVWVTVTRDTSISTAYMELSRLTSDDTAVYYC PSRFSGSGSGTDYTLTISSLARDDGSYSPFDYWGQGTMVTVSSASTKG QPEDFATYYCLQYVNLITFGPSVFPLAPCSRSTSESTAALGCLVKDYFPE GGTKVEIKRTVAAPSVFIFPPPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SDEQLKSGTASVVCLLNNFSSVVTVPSSNFGTQTYTCNVDHKPSNTKV YPREAKVQWKVDNALQSGDKTVERKSSVECPPCPAPPVAGPSVFLFPP NSQESVTEQDSKDSTYSLSKPKDTLMISRTPEVTCVVVDVSHEDPEVQF STLTLSKADYEKHKVYACEVNWYVDGVEVHNAKTKPREEQFNSTFRVVS THQGLSSPVTKSFNRGECVLTVVHQDWLNGKEYKCKVSNKGLPAPIE (SEQ ID NO: 298)KTISKTKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 302) 7 MVSSAQFLGLLLLCFQGTR MGWTLVFLFLLSVTAGVHSEVQLVQSGAE CDIQMTQSPSSLSASVGDRVKKPGASVKVSCKASGYTFTDYLMHWVRQ VTITCQASQNINKYIAWYQQAPGQGLEWMGWVDPEYGSTDYAEKFK KPGKAPKLLLYYTSTLESGVKRVTMTRDTSTSTFYMELSSLRSDDT PSRFSGSGSGTDYTLTISSLAVYFCARDDGSYSPFDYWGQGTLVTVSSA QPEDFATYYCLQYVNLITFGSTKGPSVFPLAPCSRSTSESTAALGCLVKD GGTKVEIKRTVAAPSVFIFPPYFPEPVTVSWNSGALTSGVHTFPAVLQSS SDEQLKSGTASVVCLLNNFGLYSLSSVVTVPSSNFGTQTYTCNVDHKPS YPREAKVQWKVDNALQSGNTKVDKTVERKSSVECPPCPAPPVAGPSV NSQESVTEQDSKDSTYSLSFLFPPKPKDTLMISRTPEVTCVVVDVSHED STLTLSKADYEKHKVYACEVPEVQFNWYVDGVEVHNAKTKPREEQFNST THQGLSSPVTKSFNRGECFRVVSVLTVVHQDWLNGKEYKCKVSNKGL (SEQ ID NO: 298)PAPIEKTISKTKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG K(SEQ ID NO: 303)  8 MVSSAQFLGLLLLCFQGTR MGWTLVFLFLLSVTAGVHSEVQLVESGAEVCDIQMTQSPSSLSASVGDR KKPGASVKVSCKASGYTFTDYLMHWVRQA VTITCQASQNINKYIAWYQQPGQGLEWMGWVDPEYGSTDYAEKFKK KPGKAPKLLLYYTSTLESGVRVTMTRDTSISTAYMELNRLTSDDTAVYFC PSRFSGSGSGTDYTLTISSLARDDGSYSPFDYWGQGTLVTVSSASTKGP QPEDFATYYCLQYVNLITFGSVFPLAPCSRSTSESTAALGCLVKDYFPEP GGTKVEIKRTVAAPSVFIFPPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SDEQLKSGTASVVCLLNNFSVVTVPSSNFGTQTYTCNVDHKPSNTKVD YPREAKVQWKVDNALQSGKTVERKSSVECPPCPAPPVAGPSVFLFPP NSQESVTEQDSKDSTYSLSKPKDTLMISRTPEVTCVVVDVSHEDPEVQF STLTLSKADYEKHKVYACEVNWYVDGVEVHNAKTKPREEQFNSTFRVVS THQGLSSPVTKSFNRGECVLTVVHQDWLNGKEYKCKVSNKGLPAPIEK (SEQ ID NO: 298)TISKTKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 304) 9 MVSSAQFLGLLLLCFQGTR MGWTLVFLFLLSVTAGVHSQVQLVQSGTE CDIQMTQSPSSLSASVGDRVTKPGASVKVSCKASGYTFTDYLMHWVRQ VTITCQASQNINKYIAWYQQAPGQGLEWLGWVDPEYGSTDYAEKFKKR KPGKAPKLLLYYTSTLESGVVTMTRDTSTNTVYMELTSLRSEDTAIYYCA PSRFSGSGSGTDYTLTISSLRDDGSYSPFDYWGQGTLVTVSSASTKGPS QPEDFATYYCLQYVNLITFGVFPLAPCSRSTSESTAALGCLVKDYFPEPV GGTKVEIKRTVAAPSVFIFPPTVSWNSGALTSGVHTFPAVLQSSGLYSLSS SDEQLKSGTASVVCLLNNFVVTVPSSNFGTQTYTCNVDHKPSNTKVDK YPREAKVQWKVDNALQSGTVERKSSVECPPCPAPPVAGPSVFLFPPKP NSQESVTEQDSKDSTYSLSKDTLMISRTPEVTCVVVDVSHEDPEVQFN STLTLSKADYEKHKVYACEVWYVDGVEVHNAKTKPREEQFNSTFRVVSV THQGLSSPVTKSFNRGECLTVVHQDWLNGKEYKCKVSNKGLPAPIEKT (SEQ ID NO: 298)ISKTKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 305)10 MVSSAQFLGLLLLCFQGTR MGWTLVFLFLLSVTAGVHSEVQLVQSGAE CDIQMTQSPSSLSASVGDRVKKPGATVKISCKVSGYTFTDYLMHWVQQ VTITCQASQNINKYIAWYQQAPGKGLEWMGWVDPEYGSTDYAEKFKKR KPGKAPKLLLYYTSTLESGVVTITADTSTDTAYMELSSLRSEDTAVYYCA PSRFSGSGSGTDYTLTISSLRDDGSYSPFDYWGQGVMVTVSSASTKGP QPEDFATYYCLQYVNLITFGSVFPLAPCSRSTSESTAALGCLVKDYFPEP GGTKVEIKRTVAAPSVFIFPPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SDEQLKSGTASVVCLLNNFSVVTVPSSNFGTQTYTCNVDHKPSNTKVD YPREAKVQWKVDNALQSGKTVERKSSVECPPCPAPPVAGPSVFLFPP NSQESVTEQDSKDSTYSLSKPKDTLMISRTPEVTCVVVDVSHEDPEVQF STLTLSKADYEKHKVYACEVNWYVDGVEVHNAKTKPREEQFNSTFRVVS THQGLSSPVTKSFNRGECVLTVVHQDWLNGKEYKCKVSNKGLPAPIEK (SEQ ID NO: 298)TISKTKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 306)11 MVSSAQFLGLLLLCFQGTR MGWTLVFLFLLSVTAGVHSQVQLVQSGAE CDIQMTQSPSSLSASVGDRVKKPGASVKVSCKASGYTFTDYLMHWVRQ VTITCQASQNINKYIAWYQQAPGQGLEWIGWVDPEYGSTDYAEKFKK KPGKVPTLLIFYTSTLESGVPWVTVTRDTSISTAYMELSRLTSDDTAVYYC SRFSGSGSGTDFTLTISSLQARDDGSYSPFDYWGQGTMVTVSSASTKG SEDVATYFCLQYVNLITFGGPSVFPLAPCSRSTSESTAALGCLVKDYFPE GTKVEIKRTVAAPSVFIFPSPVTVSWNSGALTSGVHTFPAVLQSSGLYSL DEQLKSGTASVVCLLNNFYSSVVTVPSSNFGTQTYTCNVDHKPSNTKV PREAKVQWKVDNALQSGNDKTVERKSSVECPPCPAPPVAGPSVFLFPP SQESVTEQDSKDSTYSLSSKPKDTLMISRTPEVTCVVVDVSHEDPEVQF TLTLSKADYEKHKVYACEVTNWYVDGVEVHNAKTKPREEQFNSTFRVVS HQGLSSPVTKSFNRGECVLTVVHQDWLNGKEYKCKVSNKGLPAPIE (SEQ ID NO: 299)KTISKTKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 302)12 MVSSAQFLGLLLLCFQGTR MGWTLVFLFLLSVTAGVHSEVQLVQSGAE CDIQMTQSPSSLSASVGDRVKKPGASVKVSCKASGYTFTDYLMHWVRQ VTITCQASQNINKYIAWYQQAPGQGLEWMGWVDPEYGSTDYAEKFK KPGKVPTLLIFYTSTLESGVPKRVTMTRDTSTSTFYMELSSLRSDDT SRFSGSGSGTDFTLTISSLQAVYFCARDDGSYSPFDYWGQGTLVTVSSA SEDVATYFCLQYVNLITFGGSTKGPSVFPLAPCSRSTSESTAALGCLVKD GTKVEIKRTVAAPSVFIFPPSYFPEPVTVSWNSGALTSGVHTFPAVLQSS DEQLKSGTASVVCLLNNFYGLYSLSSVVTVPSSNFGTQTYTCNVDHKPS PREAKVQWKVDNALQSGNNTKVDKTVERKSSVECPPCPAPPVAGPSV SQESVTEQDSKDSTYSLSSFLFPPKPKDTLMISRTPEVTCVVVDVSHED TLTLSKADYEKHKVYACEVTPEVQFNWYVDGVEVHNAKTKPREEQFNST HQGLSSPVTKSFNRGECFRVVSVLTVVHQDWLNGKEYKCKVSNKGL (SEQ ID NO: 299)PAPIEKTISKTKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG K(SEQ ID NO: 303) 13 MVSSAQFLGLLLLCFQGTR MGWTLVFLFLLSVTAGVHSEVQLVESGAEVCDIQMTQSPSSLSASVGDR KKPGASVKVSCKASGYTFTDYLMHWVRQA VTITCQASQNINKYIAWYQQPGQGLEWMGWVDPEYGSTDYAEKFKK KPGKVPTLLIFYTSTLESGVPRVTMTRDTSISTAYMELNRLTSDDTAVYFC SRFSGSGSGTDFTLTISSLQARDDGSYSPFDYWGQGTLVTVSSASTKGP SEDVATYFCLQYVNLITFGGSVFPLAPCSRSTSESTAALGCLVKDYFPEP GTKVEIKRTVAAPSVFIFPPSVTVSWNSGALTSGVHTFPAVLQSSGLYSLS DEQLKSGTASVVCLLNNFYSVVTVPSSNFGTQTYTCNVDHKPSNTKVD PREAKVQWKVDNALQSGNKTVERKSSVECPPCPAPPVAGPSVFLFPP SQESVTEQDSKDSTYSLSSKPKDTLMISRTPEVTCVVVDVSHEDPEVQF TLTLSKADYEKHKVYACEVTNWYVDGVEVHNAKTKPREEQFNSTFRVVS HQGLSSPVTKSFNRGECVLTVVHQDWLNGKEYKCKVSNKGLPAPIEK (SEQ ID NO: 299)TISKTKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 304)14 MVSSAQFLGLLLLCFQGTR MGWTLVFLFLLSVTAGVHSQVQLVQSGTE CDIQMTQSPSSLSASVGDRVTKPGASVKVSCKASGYTFTDYLMHWVRQ VTITCQASQNINKYIAWYQQAPGQGLEWLGWVDPEYGSTDYAEKFKKR KPGKVPTLLIFYTSTLESGVPVTMTRDTSTNTVYMELTSLRSEDTAIYYCA SRFSGSGSGTDFTLTISSLQRDDGSYSPFDYWGQGTLVTVSSASTKGPS SEDVATYFCLQYVNLITFGGVFPLAPCSRSTSESTAALGCLVKDYFPEPV GTKVEIKRTVAAPSVFIFPPSTVSWNSGALTSGVHTFPAVLQSSGLYSLSS DEQLKSGTASVVCLLNNFYVVTVPSSNFGTQTYTCNVDHKPSNTKVDK PREAKVQWKVDNALQSGNTVERKSSVECPPCPAPPVAGPSVFLFPPKP SQESVTEQDSKDSTYSLSSKDTLMISRTPEVTCVVVDVSHEDPEVQFN TLTLSKADYEKHKVYACEVTWYVDGVEVHNAKTKPREEQFNSTFRVVSV HQGLSSPVTKSFNRGECLTVVHQDWLNGKEYKCKVSNKGLPAPIEKT (SEQ ID NO: 299)ISKTKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 305)15 MVSSAQFLGLLLLCFQGTR MGWTLVFLFLLSVTAGVHSEVQLVQSGAE CDIQMTQSPSSLSASVGDRVKKPGATVKISCKVSGYTFTDYLMHWVQQ VTITCQASQNINKYIAWYQQAPGKGLEWMGWVDPEYGSTDYAEKFKKR KPGKVPTLLIFYTSTLESGVPVTITADTSTDTAYMELSSLRSEDTAVYYCA SRFSGSGSGTDFTLTISSLQRDDGSYSPFDYWGQGVMVTVSSASTKGP SEDVATYFCLQYVNLITFGGSVFPLAPCSRSTSESTAALGCLVKDYFPEP GTKVEIKRTVAAPSVFIFPPSVTVSWNSGALTSGVHTFPAVLQSSGLYSLS DEQLKSGTASVVCLLNNFYSVVTVPSSNFGTQTYTCNVDHKPSNTKVD PREAKVQWKVDNALQSGNKTVERKSSVECPPCPAPPVAGPSVFLFPP SQESVTEQDSKDSTYSLSSKPKDTLMISRTPEVTCVVVDVSHEDPEVQF TLTLSKADYEKHKVYACEVTNWYVDGVEVHNAKTKPREEQFNSTFRVVS HQGLSSPVTKSFNRGECVLTVVHQDWLNGKEYKCKVSNKGLPAPIEK (SEQ ID NO: 299)TISKTKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 306)16 MVSSAQFLGLLLLCFQGTR MGWTLVFLFLLSVTAGVHSQVQLVQSGAECDIQMTQSPSSLSASIGDRV VKKPGASVKVSCKASGYTFTDYLMHWVRQ TITCQASQNINKYIAWYQQKAPGQGLEWIGWVDPEYGSTDYAEKFKK PGKAPKLLIYYTSTLESGVPWVTVTRDTSISTAYMELSRLTSDDTAVYYC SRFSGSGSGTDFTFTISSLQARDDGSYSPFDYWGQGTMVTVSSASTKG PEDIGTYYCLQYVNLITFGQPSVFPLAPCSRSTSESTAALGCLVKDYFPE GTRLEIKRTVAAPSVFIFPPSPVTVSWNSGALTSGVHTFPAVLQSSGLYSL DEQLKSGTASVVCLLNNFYSSVVTVPSSNFGTQTYTCNVDHKPSNTKV PREAKVQWKVDNALQSGNDKTVERKSSVECPPCPAPPVAGPSVFLFPP SQESVTEQDSKDSTYSLSSKPKDTLMISRTPEVTCVVVDVSHEDPEVQF TLTLSKADYEKHKVYACEVTNWYVDGVEVHNAKTKPREEQFNSTFRVVS HQGLSSPVTKSFNRGECVLTVVHQDWLNGKEYKCKVSNKGLPAPIE (SEQ ID NO: 300)KTISKTKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 302)17 MVSSAQFLGLLLLCFQGTR MGWTLVFLFLLSVTAGVHSEVQLVQSGAECDIQMTQSPSSLSASIGDRV VKKPGASVKVSCKASGYTFTDYLMHWVRQ TITCQASQNINKYIAWYQQKAPGQGLEWMGWVDPEYGSTDYAEKFK PGKAPKLLIYYTSTLESGVPKRVTMTRDTSTSTFYMELSSLRSDDT SRFSGSGSGTDFTFTISSLQAVYFCARDDGSYSPFDYWGQGTLVTVSSA PEDIGTYYCLQYVNLITFGQSTKGPSVFPLAPCSRSTSESTAALGCLVKD GTRLEIKRTVAAPSVFIFPPSYFPEPVTVSWNSGALTSGVHTFPAVLQSS DEQLKSGTASVVCLLNNFYGLYSLSSVVTVPSSNFGTQTYTCNVDHKPS PREAKVQWKVDNALQSGNNTKVDKTVERKSSVECPPCPAPPVAGPSV SQESVTEQDSKDSTYSLSSFLFPPKPKDTLMISRTPEVTCVVVDVSHED TLTLSKADYEKHKVYACEVTPEVQFNWYVDGVEVHNAKTKPREEQFNST HQGLSSPVTKSFNRGECFRVVSVLTVVHQDWLNGKEYKCKVSNKGL (SEQ ID NO: 300)PAPIEKTISKTKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG K(SEQ ID NO: 303) 18 MVSSAQFLGLLLLCFQGTR MGWTLVFLFLLSVTAGVHSEVQLVESGAEVCDIQMTQSPSSLSASIGDRV KKPGASVKVSCKASGYTFTDYLMHWVRQA TITCQASQNINKYIAWYQQKPGQGLEWMGWVDPEYGSTDYAEKFKK PGKAPKLLIYYTSTLESGVPRVTMTRDTSISTAYMELNRLTSDDTAVYFC SRFSGSGSGTDFTFTISSLQARDDGSYSPFDYWGQGTLVTVSSASTKGP PEDIGTYYCLQYVNLITFGQSVFPLAPCSRSTSESTAALGCLVKDYFPEP GTRLEIKRTVAAPSVFIFPPSVTVSWNSGALTSGVHTFPAVLQSSGLYSLS DEQLKSGTASVVCLLNNFYSVVTVPSSNFGTQTYTCNVDHKPSNTKVD PREAKVQWKVDNALQSGNKTVERKSSVECPPCPAPPVAGPSVFLFPP SQESVTEQDSKDSTYSLSSKPKDTLMISRTPEVTCVVVDVSHEDPEVQF TLTLSKADYEKHKVYACEVTNWYVDGVEVHNAKTKPREEQFNSTFRVVS HQGLSSPVTKSFNRGECVLTVVHQDWLNGKEYKCKVSNKGLPAPIEK (SEQ ID NO: 300)TISKTKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 304)19 MVSSAQFLGLLLLCFQGTR MGWTLVFLFLLSVTAGVHSQVQLVQSGTECDIQMTQSPSSLSASIGDRV VTKPGASVKVSCKASGYTFTDYLMHWVRQ TITCQASQNINKYIAWYQQKAPGQGLEWLGWVDPEYGSTDYAEKFKKR PGKAPKLLIYYTSTLESGVPVTMTRDTSTNTVYMELTSLRSEDTAIYYCA SRFSGSGSGTDFTFTISSLQRDDGSYSPFDYWGQGTLVTVSSASTKGPS PEDIGTYYCLQYVNLITFGQVFPLAPCSRSTSESTAALGCLVKDYFPEPV GTRLEIKRTVAAPSVFIFPPSTVSWNSGALTSGVHTFPAVLQSSGLYSLSS DEQLKSGTASVVCLLNNFYVVTVPSSNFGTQTYTCNVDHKPSNTKVDK PREAKVQWKVDNALQSGNTVERKSSVECPPCPAPPVAGPSVFLFPPKP SQESVTEQDSKDSTYSLSSKDTLMISRTPEVTCVVVDVSHEDPEVQFN TLTLSKADYEKHKVYACEVTWYVDGVEVHNAKTKPREEQFNSTFRVVSV HQGLSSPVTKSFNRGECLTVVHQDWLNGKEYKCKVSNKGLPAPIEKT (SEQ ID NO: 300)ISKTKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 305)20 MVSSAQFLGLLLLCFQGTR MGWTLVFLFLLSVTAGVHSEVQLVQSGAECDIQMTQSPSSLSASIGDRV VKKPGATVKISCKVSGYTFTDYLMHWVQQ TITCQASQNINKYIAWYQQKAPGKGLEWMGWVDPEYGSTDYAEKFKKR PGKAPKLLIYYTSTLESGVPVTITADTSTDTAYMELSSLRSEDTAVYYCA SRFSGSGSGTDFTFTISSLQRDDGSYSPFDYWGQGVMVTVSSASTKGP PEDIGTYYCLQYVNLITFGQSVFPLAPCSRSTSESTAALGCLVKDYFPEP GTRLEIKRTVAAPSVFIFPPSVTVSWNSGALTSGVHTFPAVLQSSGLYSLS DEQLKSGTASVVCLLNNFYSVVTVPSSNFGTQTYTCNVDHKPSNTKVD PREAKVQWKVDNALQSGNKTVERKSSVECPPCPAPPVAGPSVFLFPP SQESVTEQDSKDSTYSLSSKPKDTLMISRTPEVTCVVVDVSHEDPEVQF TLTLSKADYEKHKVYACEVTNWYVDGVEVHNAKTKPREEQFNSTFRVVS HQGLSSPVTKSFNRGECVLTVVHQDWLNGKEYKCKVSNKGLPAPIEK (SEQ ID NO: 300)TISKTKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 306)21 MVSSAQFLGLLLLCFQGTR MGWTLVFLFLLSVTAGVHSQVQLVQSGAE CDIQMTQSPSSLSASVGDRVKKPGASVKVSCKASGYTFTDYLMHWVRQ VTITCQASQNINKYIAWYQQAPGQGLEWIGWVDPEYGSTDYAEKFKK KPGKAPKLLIYYTSTLESGVWVTVTRDTSISTAYMELSRLTSDDTAVYYC PSRFSGSGSGTDFTFTISSLARDDGSYSPFDYWGQGTMVTVSSASTKG QPEDIATYYCLQYVNLITFGPSVFPLAPCSRSTSESTAALGCLVKDYFPE AGTKLELKRTVAAPSVFIFPPVTVSWNSGALTSGVHTFPAVLQSSGLYSL PSDEQLKSGTASVVCLLNNSSVVTVPSSNFGTQTYTCNVDHKPSNTKV FYPREAKVQWKVDNALQSGDKTVERKSSVECPPCPAPPVAGPSVFLFPP NSQESVTEQDSKDSTYSLSKPKDTLMISRTPEVTCVVVDVSHEDPEVQF STLTLSKADYEKHKVYACEVNWYVDGVEVHNAKTKPREEQFNSTFRVVS THQGLSSPVTKSFNRGECVLTVVHQDWLNGKEYKCKVSNKGLPAPIE (SEQ ID NO: 301)KTISKTKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 302)22 MVSSAQFLGLLLLCFQGTR MGWTLVFLFLLSVTAGVHSEVQLVQSGAE CDIQMTQSPSSLSASVGDRVKKPGASVKVSCKASGYTFTDYLMHWVRQ VTITCQASQNINKYIAWYQQAPGQGLEWMGWVDPEYGSTDYAEKFK KPGKAPKLLIYYTSTLESGVKRVTMTRDTSTSTFYMELSSLRSDDT PSRFSGSGSGTDFTFTISSLAVYFCARDDGSYSPFDYWGQGTLVTVSSA QPEDIATYYCLQYVNLITFGSTKGPSVFPLAPCSRSTSESTAALGCLVKD AGTKLELKRTVAAPSVFIFPYFPEPVTVSWNSGALTSGVHTFPAVLQSS PSDEQLKSGTASVVCLLNNGLYSLSSVVTVPSSNFGTQTYTCNVDHKPS FYPREAKVQWKVDNALQSGNTKVDKTVERKSSVECPPCPAPPVAGPSV NSQESVTEQDSKDSTYSLSFLFPPKPKDTLMISRTPEVTCVVVDVSHED STLTLSKADYEKHKVYACEVPEVQFNWYVDGVEVHNAKTKPREEQFNST THQGLSSPVTKSFNRGECFRVVSVLTVVHQDWLNGKEYKCKVSNKGL (SEQ ID NO: 301)PAPIEKTISKTKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG K(SEQ ID NO: 303) 23 MVSSAQFLGLLLLCFQGTR MGWTLVFLFLLSVTAGVHSEVQLVESGAEVCDIQMTQSPSSLSASVGDR KKPGASVKVSCKASGYTFTDYLMHWVRQA VTITCQASQNINKYIAWYQQPGQGLEWMGWVDPEYGSTDYAEKFKK KPGKAPKLLIYYTSTLESGVRVTMTRDTSISTAYMELNRLTSDDTAVYFC PSRFSGSGSGTDFTFTISSLARDDGSYSPFDYWGQGTLVTVSSASTKGP QPEDIATYYCLQYVNLITFGSVFPLAPCSRSTSESTAALGCLVKDYFPEP AGTKLELKRTVAAPSVFIFPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS PSDEQLKSGTASVVCLLNNSVVTVPSSNFGTQTYTCNVDHKPSNTKVD FYPREAKVQWKVDNALQSGKTVERKSSVECPPCPAPPVAGPSVFLFPP NSQESVTEQDSKDSTYSLSKPKDTLMISRTPEVTCVVVDVSHEDPEVQF STLTLSKADYEKHKVYACEVNWYVDGVEVHNAKTKPREEQFNSTFRVVS THQGLSSPVTKSFNRGECVLTVVHQDWLNGKEYKCKVSNKGLPAPIEK (SEQ ID NO: 301)TISKTKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 304)24 MVSSAQFLGLLLLCFQGTR MGWTLVFLFLLSVTAGVHSQVQLVQSGTE CDIQMTQSPSSLSASVGDRVTKPGASVKVSCKASGYTFTDYLMHWVRQ VTITCQASQNINKYIAWYQQAPGQGLEWLGWVDPEYGSTDYAEKFKKR KPGKAPKLLIYYTSTLESGVVTMTRDTSTNTVYMELTSLRSEDTAIYYCA PSRFSGSGSGTDFTFTISSLRDDGSYSPFDYWGQGTLVTVSSASTKGPS QPEDIATYYCLQYVNLITFGVFPLAPCSRSTSESTAALGCLVKDYFPEPV AGTKLELKRTVAAPSVFIFPTVSWNSGALTSGVHTFPAVLQSSGLYSLSS PSDEQLKSGTASVVCLLNNVVTVPSSNFGTQTYTCNVDHKPSNTKVDK FYPREAKVQWKVDNALQSGTVERKSSVECPPCPAPPVAGPSVFLFPPKP NSQESVTEQDSKDSTYSLSKDTLMISRTPEVTCVVVDVSHEDPEVQFN STLTLSKADYEKHKVYACEVWYVDGVEVHNAKTKPREEQFNSTFRVVSV THQGLSSPVTKSFNRGECLTVVHQDWLNGKEYKCKVSNKGLPAPIEKT (SEQ ID NO: 301)ISKTKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 305)25 MVSSAQFLGLLLLCFQGTR MGWTLVFLFLLSVTAGVHSEVQLVQSGAE CDIQMTQSPSSLSASVGDRVKKPGATVKISCKVSGYTFTDYLMHWVQQ VTITCQASQNINKYIAWYQQAPGKGLEWMGWVDPEYGSTDYAEKFKKR KPGKAPKLLIYYTSTLESGVVTITADTSTDTAYMELSSLRSEDTAVYYCA PSRFSGSGSGTDFTFTISSLRDDGSYSPFDYWGQGVMVTVSSASTKGP QPEDIATYYCLQYVNLITFGSVFPLAPCSRSTSESTAALGCLVKDYFPEP AGTKLELKRTVAAPSVFIFPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS PSDEQLKSGTASVVCLLNNSVVTVPSSNFGTQTYTCNVDHKPSNTKVD FYPREAKVQWKVDNALQSGKTVERKSSVECPPCPAPPVAGPSVFLFPP NSQESVTEQDSKDSTYSLSKPKDTLMISRTPEVTCVVVDVSHEDPEVQF STLTLSKADYEKHKVYACEVNWYVDGVEVHNAKTKPREEQFNSTFRVVS THQGLSSPVTKSFNRGECVLTVVHQDWLNGKEYKCKVSNKGLPAPIEK (SEQ ID NO: 301)TISKTKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 306)

For example, in some embodiments, the disclosure features anantagonistic TNFR2 antibody or antigen-binding fragment thereofcontaining a heavy chain having an amino acid sequence that is at least85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to theamino acid sequence of SEQ ID NO: 302. In some embodiments, theantagonistic TNFR2 antibody or antigen-binding fragment thereof containsa heavy chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:302. In some embodiments, the antagonistic TNFR2 antibody orantigen-binding fragment thereof contains a heavy chain having an aminoacid sequence that is at least 95% identical (e.g., at least 95%, 96%,97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence ofSEQ ID NO: 302. In some embodiments, the antagonistic TNFR2 antibody orantigen-binding fragment thereof contains a heavy chain having the aminoacid sequence of SEQ ID NO: 302.

In some embodiments, the disclosure features an antagonistic TNFR2antibody or antigen-binding fragment thereof containing a heavy chainhaving an amino acid sequence that is at least 85% identical (e.g., atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ IDNO: 303. In some embodiments, the antagonistic TNFR2 antibody orantigen-binding fragment thereof contains a heavy chain having an aminoacid sequence that is at least 90% identical (e.g., at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to theamino acid sequence of SEQ ID NO: 303. In some embodiments, theantagonistic TNFR2 antibody or antigen-binding fragment thereof containsa heavy chain having an amino acid sequence that is at least 95%identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100%identical) to the amino acid sequence of SEQ ID NO: 303. In someembodiments, the antagonistic TNFR2 antibody or antigen-binding fragmentthereof contains a heavy chain having the amino acid sequence of SEQ IDNO: 303.

In some embodiments, the disclosure features an antagonistic TNFR2antibody or antigen-binding fragment thereof containing a heavy chainhaving an amino acid sequence that is at least 85% identical (e.g., atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ IDNO: 304. In some embodiments, the antagonistic TNFR2 antibody orantigen-binding fragment thereof contains a heavy chain having an aminoacid sequence that is at least 90% identical (e.g., at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to theamino acid sequence of SEQ ID NO: 304. In some embodiments, theantagonistic TNFR2 antibody or antigen-binding fragment thereof containsa heavy chain having an amino acid sequence that is at least 95%identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100%identical) to the amino acid sequence of SEQ ID NO: 304. In someembodiments, the antagonistic TNFR2 antibody or antigen-binding fragmentthereof contains a heavy chain having the amino acid sequence of SEQ IDNO: 304.

In some embodiments, the disclosure features an antagonistic TNFR2antibody or antigen-binding fragment thereof containing a heavy chainhaving an amino acid sequence that is at least 85% identical (e.g., atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ IDNO: 305. In some embodiments, the antagonistic TNFR2 antibody orantigen-binding fragment thereof contains a heavy chain having an aminoacid sequence that is at least 90% identical (e.g., at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to theamino acid sequence of SEQ ID NO: 305. In some embodiments, theantagonistic TNFR2 antibody or antigen-binding fragment thereof containsa heavy chain having an amino acid sequence that is at least 95%identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100%identical) to the amino acid sequence of SEQ ID NO: 305. In someembodiments, the antagonistic TNFR2 antibody or antigen-binding fragmentthereof contains a heavy chain having the amino acid sequence of SEQ IDNO: 305.

In some embodiments, the disclosure features an antagonistic TNFR2antibody or antigen-binding fragment thereof containing a heavy chainhaving an amino acid sequence that is at least 85% identical (e.g., atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ IDNO: 306. In some embodiments, the antagonistic TNFR2 antibody orantigen-binding fragment thereof contains a heavy chain having an aminoacid sequence that is at least 90% identical (e.g., at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to theamino acid sequence of SEQ ID NO: 306. In some embodiments, theantagonistic TNFR2 antibody or antigen-binding fragment thereof containsa heavy chain having an amino acid sequence that is at least 95%identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100%identical) to the amino acid sequence of SEQ ID NO: 306. In someembodiments, the antagonistic TNFR2 antibody or antigen-binding fragmentthereof contains a heavy chain having the amino acid sequence of SEQ IDNO: 306.

In some embodiments, the disclosure features an antagonistic TNFR2antibody or antigen-binding fragment thereof containing a light chainhaving an amino acid sequence that is at least 85% identical (e.g., atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ IDNO: 297. In some embodiments, the antagonistic TNFR2 antibody orantigen-binding fragment thereof contains a light chain having an aminoacid sequence that is at least 90% identical (e.g., at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to theamino acid sequence of SEQ ID NO: 297. In some embodiments, theantagonistic TNFR2 antibody or antigen-binding fragment thereof containsa light chain having an amino acid sequence that is at least 95%identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100%identical) to the amino acid sequence of SEQ ID NO: 297. In someembodiments, the antagonistic TNFR2 antibody or antigen-binding fragmentthereof contains a light chain having the amino acid sequence of SEQ IDNO: 297.

In some embodiments, the disclosure features an antagonistic TNFR2antibody or antigen-binding fragment thereof containing a light chainhaving an amino acid sequence that is at least 85% identical (e.g., atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ IDNO: 298. In some embodiments, the antagonistic TNFR2 antibody orantigen-binding fragment thereof contains a light chain having an aminoacid sequence that is at least 90% identical (e.g., at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to theamino acid sequence of SEQ ID NO: 298. In some embodiments, theantagonistic TNFR2 antibody or antigen-binding fragment thereof containsa light chain having an amino acid sequence that is at least 95%identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100%identical) to the amino acid sequence of SEQ ID NO: 298. In someembodiments, the antagonistic TNFR2 antibody or antigen-binding fragmentthereof contains a light chain having the amino acid sequence of SEQ IDNO: 298.

In some embodiments, the disclosure features an antagonistic TNFR2antibody or antigen-binding fragment thereof containing a light chainhaving an amino acid sequence that is at least 85% identical (e.g., atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ IDNO: 299. In some embodiments, the antagonistic TNFR2 antibody orantigen-binding fragment thereof contains a light chain having an aminoacid sequence that is at least 90% identical (e.g., at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to theamino acid sequence of SEQ ID NO: 299. In some embodiments, theantagonistic TNFR2 antibody or antigen-binding fragment thereof containsa light chain having an amino acid sequence that is at least 95%identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100%identical) to the amino acid sequence of SEQ ID NO: 299. In someembodiments, the antagonistic TNFR2 antibody or antigen-binding fragmentthereof contains a light chain having the amino acid sequence of SEQ IDNO: 299.

In some embodiments, the disclosure features an antagonistic TNFR2antibody or antigen-binding fragment thereof containing a light chainhaving an amino acid sequence that is at least 85% identical (e.g., atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ IDNO: 300. In some embodiments, the antagonistic TNFR2 antibody orantigen-binding fragment thereof contains a light chain having an aminoacid sequence that is at least 90% identical (e.g., at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to theamino acid sequence of SEQ ID NO: 300. In some embodiments, theantagonistic TNFR2 antibody or antigen-binding fragment thereof containsa light chain having an amino acid sequence that is at least 95%identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100%identical) to the amino acid sequence of SEQ ID NO: 300. In someembodiments, the antagonistic TNFR2 antibody or antigen-binding fragmentthereof contains a light chain having the amino acid sequence of SEQ IDNO: 300.

In some embodiments, the disclosure features an antagonistic TNFR2antibody or antigen-binding fragment thereof containing a light chainhaving an amino acid sequence that is at least 85% identical (e.g., atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ IDNO: 301. In some embodiments, the antagonistic TNFR2 antibody orantigen-binding fragment thereof contains a light chain having an aminoacid sequence that is at least 90% identical (e.g., at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to theamino acid sequence of SEQ ID NO: 301. In some embodiments, theantagonistic TNFR2 antibody or antigen-binding fragment thereof containsa light chain having an amino acid sequence that is at least 95%identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100%identical) to the amino acid sequence of SEQ ID NO: 301. In someembodiments, the antagonistic TNFR2 antibody or antigen-binding fragmentthereof contains a light chain having the amino acid sequence of SEQ IDNO: 301.

In some embodiments, the disclosure features an antagonistic TNFR2antibody or antigen-binding fragment thereof containing a heavy chainhaving an amino acid sequence that is at least 85% identical (e.g., atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ IDNO: 302 and a light chain having an amino acid sequence that is at least85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to theamino acid sequence of SEQ ID NO: 297. In some embodiments, theantagonistic TNFR2 antibody of antigen-binding fragment thereof containsa heavy chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:302 and a light chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:297. In some embodiments, the antagonistic TNFR2 antibody ofantigen-binding fragment thereof contains a heavy chain having an aminoacid sequence that is at least 95% identical (e.g., at least 95%, 96%,97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence ofSEQ ID NO: 302 and a light chain having an amino acid sequence that isat least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%,or 100% identical) to the amino acid sequence of SEQ ID NO: 297. In someembodiments, the antagonistic TNFR2 antibody of antigen-binding fragmentthereof contains a heavy chain having the amino acid sequence of SEQ IDNO: 302 and a light chain having the amino acid sequence of SEQ ID NO:297.

In some embodiments, the disclosure features an antagonistic TNFR2antibody or antigen-binding fragment thereof containing a heavy chainhaving an amino acid sequence that is at least 85% identical (e.g., atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ IDNO: 302 and a light chain having an amino acid sequence that is at least85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to theamino acid sequence of SEQ ID NO: 298. In some embodiments, theantagonistic TNFR2 antibody of antigen-binding fragment thereof containsa heavy chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:302 and a light chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:298. In some embodiments, the antagonistic TNFR2 antibody ofantigen-binding fragment thereof contains a heavy chain having an aminoacid sequence that is at least 95% identical (e.g., at least 95%, 96%,97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence ofSEQ ID NO: 302 and a light chain having an amino acid sequence that isat least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%,or 100% identical) to the amino acid sequence of SEQ ID NO: 298. In someembodiments, the antagonistic TNFR2 antibody of antigen-binding fragmentthereof contains a heavy chain having the amino acid sequence of SEQ IDNO: 302 and a light chain having the amino acid sequence of SEQ ID NO:298.

In some embodiments, the disclosure features an antagonistic TNFR2antibody or antigen-binding fragment thereof containing a heavy chainhaving an amino acid sequence that is at least 85% identical (e.g., atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ IDNO: 302 and a light chain having an amino acid sequence that is at least85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to theamino acid sequence of SEQ ID NO: 299. In some embodiments, theantagonistic TNFR2 antibody of antigen-binding fragment thereof containsa heavy chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:302 and a light chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:299. In some embodiments, the antagonistic TNFR2 antibody ofantigen-binding fragment thereof contains a heavy chain having an aminoacid sequence that is at least 95% identical (e.g., at least 95%, 96%,97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence ofSEQ ID NO: 302 and a light chain having an amino acid sequence that isat least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%,or 100% identical) to the amino acid sequence of SEQ ID NO: 299. In someembodiments, the antagonistic TNFR2 antibody of antigen-binding fragmentthereof contains a heavy chain having the amino acid sequence of SEQ IDNO: 302 and a light chain having the amino acid sequence of SEQ ID NO:299.

In some embodiments, the disclosure features an antagonistic TNFR2antibody or antigen-binding fragment thereof containing a heavy chainhaving an amino acid sequence that is at least 85% identical (e.g., atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ IDNO: 302 and a light chain having an amino acid sequence that is at least85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to theamino acid sequence of SEQ ID NO: 300. In some embodiments, theantagonistic TNFR2 antibody of antigen-binding fragment thereof containsa heavy chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:302 and a light chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:300. In some embodiments, the antagonistic TNFR2 antibody ofantigen-binding fragment thereof contains a heavy chain having an aminoacid sequence that is at least 95% identical (e.g., at least 95%, 96%,97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence ofSEQ ID NO: 302 and a light chain having an amino acid sequence that isat least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%,or 100% identical) to the amino acid sequence of SEQ ID NO: 300. In someembodiments, the antagonistic TNFR2 antibody of antigen-binding fragmentthereof contains a heavy chain having the amino acid sequence of SEQ IDNO: 302 and a light chain having the amino acid sequence of SEQ ID NO:300.

In some embodiments, the disclosure features an antagonistic TNFR2antibody or antigen-binding fragment thereof containing a heavy chainhaving an amino acid sequence that is at least 85% identical (e.g., atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ IDNO: 302 and a light chain having an amino acid sequence that is at least85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to theamino acid sequence of SEQ ID NO: 301. In some embodiments, theantagonistic TNFR2 antibody of antigen-binding fragment thereof containsa heavy chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:302 and a light chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:301. In some embodiments, the antagonistic TNFR2 antibody ofantigen-binding fragment thereof contains a heavy chain having an aminoacid sequence that is at least 95% identical (e.g., at least 95%, 96%,97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence ofSEQ ID NO: 302 and a light chain having an amino acid sequence that isat least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%,or 100% identical) to the amino acid sequence of SEQ ID NO: 301. In someembodiments, the antagonistic TNFR2 antibody of antigen-binding fragmentthereof contains a heavy chain having the amino acid sequence of SEQ IDNO: 302 and a light chain having the amino acid sequence of SEQ ID NO:301.

In some embodiments, the disclosure features an antagonistic TNFR2antibody or antigen-binding fragment thereof containing a heavy chainhaving an amino acid sequence that is at least 85% identical (e.g., atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ IDNO: 303 and a light chain having an amino acid sequence that is at least85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to theamino acid sequence of SEQ ID NO: 297. In some embodiments, theantagonistic TNFR2 antibody of antigen-binding fragment thereof containsa heavy chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:303 and a light chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:297. In some embodiments, the antagonistic TNFR2 antibody ofantigen-binding fragment thereof contains a heavy chain having an aminoacid sequence that is at least 95% identical (e.g., at least 95%, 96%,97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence ofSEQ ID NO: 303 and a light chain having an amino acid sequence that isat least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%,or 100% identical) to the amino acid sequence of SEQ ID NO: 297. In someembodiments, the antagonistic TNFR2 antibody of antigen-binding fragmentthereof contains a heavy chain having the amino acid sequence of SEQ IDNO: 303 and a light chain having the amino acid sequence of SEQ ID NO:297.

In some embodiments, the disclosure features an antagonistic TNFR2antibody or antigen-binding fragment thereof containing a heavy chainhaving an amino acid sequence that is at least 85% identical (e.g., atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ IDNO: 303 and a light chain having an amino acid sequence that is at least85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to theamino acid sequence of SEQ ID NO: 298. In some embodiments, theantagonistic TNFR2 antibody of antigen-binding fragment thereof containsa heavy chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:303 and a light chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:298. In some embodiments, the antagonistic TNFR2 antibody ofantigen-binding fragment thereof contains a heavy chain having an aminoacid sequence that is at least 95% identical (e.g., at least 95%, 96%,97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence ofSEQ ID NO: 303 and a light chain having an amino acid sequence that isat least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%,or 100% identical) to the amino acid sequence of SEQ ID NO: 298. In someembodiments, the antagonistic TNFR2 antibody of antigen-binding fragmentthereof contains a heavy chain having the amino acid sequence of SEQ IDNO: 303 and a light chain having the amino acid sequence of SEQ ID NO:298.

In some embodiments, the disclosure features an antagonistic TNFR2antibody or antigen-binding fragment thereof containing a heavy chainhaving an amino acid sequence that is at least 85% identical (e.g., atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ IDNO: 303 and a light chain having an amino acid sequence that is at least85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to theamino acid sequence of SEQ ID NO: 299. In some embodiments, theantagonistic TNFR2 antibody of antigen-binding fragment thereof containsa heavy chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:303 and a light chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:299. In some embodiments, the antagonistic TNFR2 antibody ofantigen-binding fragment thereof contains a heavy chain having an aminoacid sequence that is at least 95% identical (e.g., at least 95%, 96%,97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence ofSEQ ID NO: 303 and a light chain having an amino acid sequence that isat least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%,or 100% identical) to the amino acid sequence of SEQ ID NO: 299. In someembodiments, the antagonistic TNFR2 antibody of antigen-binding fragmentthereof contains a heavy chain having the amino acid sequence of SEQ IDNO: 303 and a light chain having the amino acid sequence of SEQ ID NO:299.

In some embodiments, the disclosure features an antagonistic TNFR2antibody or antigen-binding fragment thereof containing a heavy chainhaving an amino acid sequence that is at least 85% identical (e.g., atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ IDNO: 303 and a light chain having an amino acid sequence that is at least85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to theamino acid sequence of SEQ ID NO: 300. In some embodiments, theantagonistic TNFR2 antibody of antigen-binding fragment thereof containsa heavy chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:303 and a light chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:300. In some embodiments, the antagonistic TNFR2 antibody ofantigen-binding fragment thereof contains a heavy chain having an aminoacid sequence that is at least 95% identical (e.g., at least 95%, 96%,97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence ofSEQ ID NO: 303 and a light chain having an amino acid sequence that isat least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%,or 100% identical) to the amino acid sequence of SEQ ID NO: 300. In someembodiments, the antagonistic TNFR2 antibody of antigen-binding fragmentthereof contains a heavy chain having the amino acid sequence of SEQ IDNO: 303 and a light chain having the amino acid sequence of SEQ ID NO:300.

In some embodiments, the disclosure features an antagonistic TNFR2antibody or antigen-binding fragment thereof containing a heavy chainhaving an amino acid sequence that is at least 85% identical (e.g., atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ IDNO: 303 and a light chain having an amino acid sequence that is at least85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to theamino acid sequence of SEQ ID NO: 301. In some embodiments, theantagonistic TNFR2 antibody of antigen-binding fragment thereof containsa heavy chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:303 and a light chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:301. In some embodiments, the antagonistic TNFR2 antibody ofantigen-binding fragment thereof contains a heavy chain having an aminoacid sequence that is at least 95% identical (e.g., at least 95%, 96%,97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence ofSEQ ID NO: 303 and a light chain having an amino acid sequence that isat least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%,or 100% identical) to the amino acid sequence of SEQ ID NO: 301. In someembodiments, the antagonistic TNFR2 antibody of antigen-binding fragmentthereof contains a heavy chain having the amino acid sequence of SEQ IDNO: 303 and a light chain having the amino acid sequence of SEQ ID NO:301.

In some embodiments, the disclosure features an antagonistic TNFR2antibody or antigen-binding fragment thereof containing a heavy chainhaving an amino acid sequence that is at least 85% identical (e.g., atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ IDNO: 304 and a light chain having an amino acid sequence that is at least85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to theamino acid sequence of SEQ ID NO: 297. In some embodiments, theantagonistic TNFR2 antibody of antigen-binding fragment thereof containsa heavy chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:304 and a light chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:297. In some embodiments, the antagonistic TNFR2 antibody ofantigen-binding fragment thereof contains a heavy chain having an aminoacid sequence that is at least 95% identical (e.g., at least 95%, 96%,97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence ofSEQ ID NO: 304 and a light chain having an amino acid sequence that isat least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%,or 100% identical) to the amino acid sequence of SEQ ID NO: 297. In someembodiments, the antagonistic TNFR2 antibody of antigen-binding fragmentthereof contains a heavy chain having the amino acid sequence of SEQ IDNO: 304 and a light chain having the amino acid sequence of SEQ ID NO:297.

In some embodiments, the disclosure features an antagonistic TNFR2antibody or antigen-binding fragment thereof containing a heavy chainhaving an amino acid sequence that is at least 85% identical (e.g., atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ IDNO: 304 and a light chain having an amino acid sequence that is at least85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to theamino acid sequence of SEQ ID NO: 298. In some embodiments, theantagonistic TNFR2 antibody of antigen-binding fragment thereof containsa heavy chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:304 and a light chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:298. In some embodiments, the antagonistic TNFR2 antibody ofantigen-binding fragment thereof contains a heavy chain having an aminoacid sequence that is at least 95% identical (e.g., at least 95%, 96%,97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence ofSEQ ID NO: 304 and a light chain having an amino acid sequence that isat least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%,or 100% identical) to the amino acid sequence of SEQ ID NO: 298. In someembodiments, the antagonistic TNFR2 antibody of antigen-binding fragmentthereof contains a heavy chain having the amino acid sequence of SEQ IDNO: 304 and a light chain having the amino acid sequence of SEQ ID NO:298.

In some embodiments, the disclosure features an antagonistic TNFR2antibody or antigen-binding fragment thereof containing a heavy chainhaving an amino acid sequence that is at least 85% identical (e.g., atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ IDNO: 304 and a light chain having an amino acid sequence that is at least85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to theamino acid sequence of SEQ ID NO: 299. In some embodiments, theantagonistic TNFR2 antibody of antigen-binding fragment thereof containsa heavy chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:304 and a light chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:299. In some embodiments, the antagonistic TNFR2 antibody ofantigen-binding fragment thereof contains a heavy chain having an aminoacid sequence that is at least 95% identical (e.g., at least 95%, 96%,97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence ofSEQ ID NO: 304 and a light chain having an amino acid sequence that isat least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%,or 100% identical) to the amino acid sequence of SEQ ID NO: 299. In someembodiments, the antagonistic TNFR2 antibody of antigen-binding fragmentthereof contains a heavy chain having the amino acid sequence of SEQ IDNO: 304 and a light chain having the amino acid sequence of SEQ ID NO:299.

In some embodiments, the disclosure features an antagonistic TNFR2antibody or antigen-binding fragment thereof containing a heavy chainhaving an amino acid sequence that is at least 85% identical (e.g., atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ IDNO: 304 and a light chain having an amino acid sequence that is at least85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to theamino acid sequence of SEQ ID NO: 300. In some embodiments, theantagonistic TNFR2 antibody of antigen-binding fragment thereof containsa heavy chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:304 and a light chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:300. In some embodiments, the antagonistic TNFR2 antibody ofantigen-binding fragment thereof contains a heavy chain having an aminoacid sequence that is at least 95% identical (e.g., at least 95%, 96%,97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence ofSEQ ID NO: 304 and a light chain having an amino acid sequence that isat least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%,or 100% identical) to the amino acid sequence of SEQ ID NO: 300. In someembodiments, the antagonistic TNFR2 antibody of antigen-binding fragmentthereof contains a heavy chain having the amino acid sequence of SEQ IDNO: 304 and a light chain having the amino acid sequence of SEQ ID NO:300.

In some embodiments, the disclosure features an antagonistic TNFR2antibody or antigen-binding fragment thereof containing a heavy chainhaving an amino acid sequence that is at least 85% identical (e.g., atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ IDNO: 304 and a light chain having an amino acid sequence that is at least85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to theamino acid sequence of SEQ ID NO: 301. In some embodiments, theantagonistic TNFR2 antibody of antigen-binding fragment thereof containsa heavy chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:304 and a light chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:301. In some embodiments, the antagonistic TNFR2 antibody ofantigen-binding fragment thereof contains a heavy chain having an aminoacid sequence that is at least 95% identical (e.g., at least 95%, 96%,97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence ofSEQ ID NO: 304 and a light chain having an amino acid sequence that isat least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%,or 100% identical) to the amino acid sequence of SEQ ID NO: 301. In someembodiments, the antagonistic TNFR2 antibody of antigen-binding fragmentthereof contains a heavy chain having the amino acid sequence of SEQ IDNO: 304 and a light chain having the amino acid sequence of SEQ ID NO:301.

In some embodiments, the disclosure features an antagonistic TNFR2antibody or antigen-binding fragment thereof containing a heavy chainhaving an amino acid sequence that is at least 85% identical (e.g., atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ IDNO: 305 and a light chain having an amino acid sequence that is at least85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to theamino acid sequence of SEQ ID NO: 297. In some embodiments, theantagonistic TNFR2 antibody of antigen-binding fragment thereof containsa heavy chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:305 and a light chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:297. In some embodiments, the antagonistic TNFR2 antibody ofantigen-binding fragment thereof contains a heavy chain having an aminoacid sequence that is at least 95% identical (e.g., at least 95%, 96%,97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence ofSEQ ID NO: 305 and a light chain having an amino acid sequence that isat least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%,or 100% identical) to the amino acid sequence of SEQ ID NO: 297. In someembodiments, the antagonistic TNFR2 antibody of antigen-binding fragmentthereof contains a heavy chain having the amino acid sequence of SEQ IDNO: 305 and a light chain having the amino acid sequence of SEQ ID NO:297.

In some embodiments, the disclosure features an antagonistic TNFR2antibody or antigen-binding fragment thereof containing heavy chainhaving an amino acid sequence that is at least 85% identical (e.g., atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ IDNO: 305 and a light chain having an amino acid sequence that is at least85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to theamino acid sequence of SEQ ID NO: 298. In some embodiments, theantagonistic TNFR2 antibody of antigen-binding fragment thereof containsa heavy chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:305 and a light chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:298. In some embodiments, the antagonistic TNFR2 antibody ofantigen-binding fragment thereof contains a heavy chain having an aminoacid sequence that is at least 95% identical (e.g., at least 95%, 96%,97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence ofSEQ ID NO: 305 and a light chain having an amino acid sequence that isat least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%,or 100% identical) to the amino acid sequence of SEQ ID NO: 298. In someembodiments, the antagonistic TNFR2 antibody of antigen-binding fragmentthereof contains a heavy chain having the amino acid sequence of SEQ IDNO: 305 and a light chain having the amino acid sequence of SEQ ID NO:298.

In some embodiments, the disclosure features an antagonistic TNFR2antibody or antigen-binding fragment thereof containing a heavy chainhaving an amino acid sequence that is at least 85% identical (e.g., atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ IDNO: 305 and a light chain having an amino acid sequence that is at least85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to theamino acid sequence of SEQ ID NO: 299. In some embodiments, theantagonistic TNFR2 antibody of antigen-binding fragment thereof containsa heavy chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:305 and a light chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:299. In some embodiments, the antagonistic TNFR2 antibody ofantigen-binding fragment thereof contains a heavy chain having an aminoacid sequence that is at least 95% identical (e.g., at least 95%, 96%,97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence ofSEQ ID NO: 305 and a light chain having an amino acid sequence that isat least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%,or 100% identical) to the amino acid sequence of SEQ ID NO: 299. In someembodiments, the antagonistic TNFR2 antibody of antigen-binding fragmentthereof contains a heavy chain having the amino acid sequence of SEQ IDNO: 305 and a light chain having the amino acid sequence of SEQ ID NO:299.

In some embodiments, the disclosure features an antagonistic TNFR2antibody or antigen-binding fragment thereof containing a heavy chainhaving an amino acid sequence that is at least 85% identical (e.g., atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ IDNO: 305 and a light chain having an amino acid sequence that is at least85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to theamino acid sequence of SEQ ID NO: 300. In some embodiments, theantagonistic TNFR2 antibody of antigen-binding fragment thereof containsa heavy chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:305 and a light chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:300. In some embodiments, the antagonistic TNFR2 antibody ofantigen-binding fragment thereof contains a heavy chain having an aminoacid sequence that is at least 95% identical (e.g., at least 95%, 96%,97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence ofSEQ ID NO: 305 and a light chain having an amino acid sequence that isat least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%,or 100% identical) to the amino acid sequence of SEQ ID NO: 300. In someembodiments, the antagonistic TNFR2 antibody of antigen-binding fragmentthereof contains a heavy chain having the amino acid sequence of SEQ IDNO: 305 and a light chain having the amino acid sequence of SEQ ID NO:300.

In some embodiments, the disclosure features an antagonistic TNFR2antibody or antigen-binding fragment thereof containing a heavy chainhaving an amino acid sequence that is at least 85% identical (e.g., atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ IDNO: 305 and a light chain having an amino acid sequence that is at least85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to theamino acid sequence of SEQ ID NO: 301. In some embodiments, theantagonistic TNFR2 antibody of antigen-binding fragment thereof containsa heavy chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:305 and a light chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:301. In some embodiments, the antagonistic TNFR2 antibody ofantigen-binding fragment thereof contains a heavy chain having an aminoacid sequence that is at least 95% identical (e.g., at least 95%, 96%,97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence ofSEQ ID NO: 305 and a light chain having an amino acid sequence that isat least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%,or 100% identical) to the amino acid sequence of SEQ ID NO: 301. In someembodiments, the antagonistic TNFR2 antibody of antigen-binding fragmentthereof contains a heavy chain having the amino acid sequence of SEQ IDNO: 305 and a light chain having the amino acid sequence of SEQ ID NO:301.

In some embodiments, the disclosure features an antagonistic TNFR2antibody or antigen-binding fragment thereof containing a heavy chainhaving an amino acid sequence that is at least 85% identical (e.g., atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ IDNO: 306 and a light chain having an amino acid sequence that is at least85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to theamino acid sequence of SEQ ID NO: 297. In some embodiments, theantagonistic TNFR2 antibody of antigen-binding fragment thereof containsa heavy chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:306 and a light chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:297. In some embodiments, the antagonistic TNFR2 antibody ofantigen-binding fragment thereof contains a heavy chain having an aminoacid sequence that is at least 95% identical (e.g., at least 95%, 96%,97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence ofSEQ ID NO: 306 and a light chain having an amino acid sequence that isat least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%,or 100% identical) to the amino acid sequence of SEQ ID NO: 297. In someembodiments, the antagonistic TNFR2 antibody of antigen-binding fragmentthereof contains a heavy chain having the amino acid sequence of SEQ IDNO: 306 and a light chain having the amino acid sequence of SEQ ID NO:297.

In some embodiments, the disclosure features an antagonistic TNFR2antibody or antigen-binding fragment thereof containing a heavy chainhaving an amino acid sequence that is at least 85% identical (e.g., atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ IDNO: 306 and a light chain having an amino acid sequence that is at least85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to theamino acid sequence of SEQ ID NO: 298. In some embodiments, theantagonistic TNFR2 antibody of antigen-binding fragment thereof containsa heavy chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:306 and a light chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:298. In some embodiments, the antagonistic TNFR2 antibody ofantigen-binding fragment thereof contains a heavy chain having an aminoacid sequence that is at least 95% identical (e.g., at least 95%, 96%,97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence ofSEQ ID NO: 306 and a light chain having an amino acid sequence that isat least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%,or 100% identical) to the amino acid sequence of SEQ ID NO: 298. In someembodiments, the antagonistic TNFR2 antibody of antigen-binding fragmentthereof contains a heavy chain having the amino acid sequence of SEQ IDNO: 306 and a light chain having the amino acid sequence of SEQ ID NO:298.

In some embodiments, the disclosure features an antagonistic TNFR2antibody or antigen-binding fragment thereof containing a heavy chainhaving an amino acid sequence that is at least 85% identical (e.g., atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ IDNO: 306 and a light chain having an amino acid sequence that is at least85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to theamino acid sequence of SEQ ID NO: 299. In some embodiments, theantagonistic TNFR2 antibody of antigen-binding fragment thereof containsa heavy chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:306 and a light chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:299. In some embodiments, the antagonistic TNFR2 antibody ofantigen-binding fragment thereof contains a heavy chain having an aminoacid sequence that is at least 95% identical (e.g., at least 95%, 96%,97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence ofSEQ ID NO: 306 and a light chain having an amino acid sequence that isat least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%,or 100% identical) to the amino acid sequence of SEQ ID NO: 299. In someembodiments, the antagonistic TNFR2 antibody of antigen-binding fragmentthereof contains a heavy chain having the amino acid sequence of SEQ IDNO: 306 and a light chain having the amino acid sequence of SEQ ID NO:299.

In some embodiments, the disclosure features an antagonistic TNFR2antibody or antigen-binding fragment thereof containing a heavy chainhaving an amino acid sequence that is at least 85% identical (e.g., atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ IDNO: 306 and a light chain having an amino acid sequence that is at least85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to theamino acid sequence of SEQ ID NO: 300. In some embodiments, theantagonistic TNFR2 antibody of antigen-binding fragment thereof containsa heavy chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:306 and a light chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:300. In some embodiments, the antagonistic TNFR2 antibody ofantigen-binding fragment thereof contains a heavy chain having an aminoacid sequence that is at least 95% identical (e.g., at least 95%, 96%,97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence ofSEQ ID NO: 306 and a light chain having an amino acid sequence that isat least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%,or 100% identical) to the amino acid sequence of SEQ ID NO: 300. In someembodiments, the antagonistic TNFR2 antibody of antigen-binding fragmentthereof contains a heavy chain having the amino acid sequence of SEQ IDNO: 306 and a light chain having the amino acid sequence of SEQ ID NO:300.

In some embodiments, the disclosure features an antagonistic TNFR2antibody or antigen-binding fragment thereof containing a heavy chainhaving an amino acid sequence that is at least 85% identical (e.g., atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ IDNO: 306 and a light chain having an amino acid sequence that is at least85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to theamino acid sequence of SEQ ID NO: 301. In some embodiments, theantagonistic TNFR2 antibody of antigen-binding fragment thereof containsa heavy chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:306 and a light chain having an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:301. In some embodiments, the antagonistic TNFR2 antibody ofantigen-binding fragment thereof contains a heavy chain having an aminoacid sequence that is at least 95% identical (e.g., at least 95%, 96%,97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence ofSEQ ID NO: 306 and a light chain having an amino acid sequence that isat least 95% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.9%,or 100% identical) to the amino acid sequence of SEQ ID NO: 301. In someembodiments, the antagonistic TNFR2 antibody of antigen-binding fragmentthereof contains a heavy chain having the amino acid sequence of SEQ IDNO: 306 and a light chain having the amino acid sequence of SEQ ID NO:301.

In some embodiments of the disclosure, the polypeptides, such assingle-chain polypeptides, antibodies, antigen-binding fragmentsthereof, and constructs thereof, contain a human IgG2 hinge region thatlacks a cysteine residue at positions 232 and/or 233 of the amino acidsequence of the IgG2 hinge region. For example, the polypeptide (e.g., asingle-chain polypeptide, antibody, antigen-binding fragment thereof, orconstruct thereof) may contain a human IgG2 hinge region having an aminoacid other than cysteine, such as a serine residue, at positions 232and/or 233 of the amino acid sequence of the IgG2 hinge region.

The polypeptide (e.g., a single-chain polypeptide, antibody,antigen-binding fragment thereof, or construct thereof) may contain, forexample, a human IgG2 hinge region having an amino acid substitution ordeletion at one or both of cysteine residues 232 and 233. The amino acidsubstitution may be a conservative amino acid substitution, such as aC232S and/or C233S amino acid substitution.

In some embodiments, the IgG2 hinge region has an amino acid sequencethat is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100%identical) to the amino acid sequence of SEQ ID NO: 291, e.g., providedthat the IgG2 hinge region contains serine residues at one or both ofpositions 232 and 233 of the IgG2 hinge amino acid sequence. The IgG2hinge region may have, for example, an amino acid sequence that is atleast 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence ofSEQ ID NO: 291, e.g., provided that the IgG2 hinge region containsserine residues at positions 232 and 233 of the IgG2 hinge amino acidsequence. In some embodiments, the IgG2 hinge region has an amino acidsequence that is at least 95% identical (e.g., at least 95%, 96%, 97%,98%, 99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ IDNO: 291, e.g., provided that the IgG2 hinge region contains serineresidues at positions 232 and 233 of the IgG2 hinge amino acid sequence.

The polypeptide (e.g., a single-chain polypeptide, antibody,antigen-binding fragment thereof, or construct thereof) may containantigen-binding sites that are separated from one another by a distanceof at least about 133 Å (e.g., by a distance of from about 133 Å toabout 160 Å, such as a distance of about 133 Å, 134 Å, 135 Å, 136 Å, 137Å, 138 Å, 139 Å, 140 Å, 141 Å, 142 Å, 143 Å, 144 Å, 145 Å, 146 Å, 147 Å,148 Å, 149 Å, 150 Å, 151 Å, 152 Å, 153 Å, 154 Å, 155 Å, 156 Å, 157 Å,158 Å, 159 Å, or 160 Å). In some embodiments, the antigen-binding sitesare separated from one another by a distance of at least about 134 Å(e.g., by a distance of from about 134 Å to about 160 Å, such as adistance of about 134 Å, 135 Å, 136 Å, 137 Å, 138 Å, 139 Å, 140 Å, 141Å, 142 Å, 143 Å, 144 Å, 145 Å, 146 Å, 147 Å, 148 Å, 149 Å, 150 Å, 151 Å,152 Å, 153 Å, 154 Å, 155 Å, 156 Å, 157 Å, 158 Å, 159 Å, or 160 Å). Insome embodiments, the antigen-binding sites are separated from oneanother by a distance of at least about 139 Å (e.g., by a distance offrom about 139 Å to about 160 Å, such as a distance of about 139 Å, 140Å, 141 Å, 142 Å, 143 Å, 144 Å, 145 Å, 146 Å, 147 Å, 148 Å, 149 Å, 150 Å,151 Å, 152 Å, 153 Å, 154 Å, 155 Å, 156 Å, 157 Å, 158 Å, 159 Å, or 160Å). In some embodiments, the antigen-binding sites are separated fromone another by a distance of at least about 150 Å (e.g., by a distanceof from about 150 Å to about 160 Å, such as a distance of about 150 Å,151 Å, 152 Å, 153 Å, 154 Å, 155 Å, 156 Å, 157 Å, 158 Å, 159 Å, or 160Å).

For example, the polypeptide (e.g., a single-chain polypeptide,antibody, antigen-binding fragment thereof, or construct thereof) maycontain antigen-binding sites that are separated from one another by adistance of from about 133 Å to about 150 Å, such as by a distance ofabout 133 Å, 134 Å, 135 Å, 136 Å, 137 Å, 138 Å, 139 Å, 140 Å, 141 Å, 142Å, 143 Å, 144 Å, 145 Å, 146 Å, 147 Å, 148 Å, 149 Å, or 150 Å. In someembodiments, the antigen-binding are separated from one another by adistance of from about 133 Å to about 145 Å, such as by a distance ofabout 133 Å, 134 Å, 135 Å, 136 Å, 137 Å, 138 Å, 139 Å, 140 Å, 141 Å, 142Å, 143 Å, 144 Å, or 145 Å. In some embodiments, the antigen-binding areseparated from one another by a distance of from about 133 Å to about139 Å, such as by a distance of about 133 Å, 134 Å, 135 Å, 136 Å, 137 Å,138 Å, or 139 Å. In some embodiments, the antigen-binding are separatedfrom one another by a distance of from about 134 Å to about 139 Å, suchas by a distance of about 134 Å, 135 Å, 136 Å, 137 Å, 138 Å, or 139 Å.

The polypeptide (e.g., a single-chain polypeptide, antibody,antigen-binding fragment thereof, or construct thereof) may contain acomplementarity-determining region (CDR) heavy chain 1 (CDR1) having theamino acid sequence GJTF(J)₂Y (SEQ ID NO: 276) or GJTF(J)₂YJ (SEQ ID NO:277), in which each J is independently a naturally occurring amino acid.In some embodiments, the polypeptide (e.g., a single-chain polypeptide,antibody, antigen-binding fragment thereof, or construct thereof)further contains:

-   -   (a) a CDR-H2 having the amino acid sequence (J)₃GSJ or (J)₅GSJ;    -   (b) a CDR-H3 having the amino acid sequence JRJDGJSJY(J)₂FDJ        (SEQ ID NO: 278) or JRJDGSY(J)₂FD(J)₃ (SEQ ID NO: 279);    -   (c) a CDR-L1 having the amino acid sequence (J)₉Y or (J)₅Y;    -   (d) a CDR-L2 having the amino acid sequence (J)₆S or (J)₂S;        and/or    -   (e) a CDR-L3 having the amino acid sequence (J)₅Y(J)₂T or        (J)₃Y(J)₄T,    -   in which each J is independently a naturally occurring amino        acid.

The polypeptide (e.g., a single-chain polypeptide, antibody,antigen-binding fragment thereof, or construct thereof) may contain aCDR-H1 having the amino acid sequence Z⁴FZ³Z⁵SSZ⁵ or Z⁴YZ³Z⁵TDZ⁵X;

-   -   In which each Z³ is independently an amino acid including a        polar, uncharged side-chain at physiological pH;    -   each Z⁴ is independently a glycine or alanine;    -   each Z⁵ is independently an amino acid including a hydrophobic        side-chain; and    -   each X is independently leucine or isoleucine.

In some embodiments, the polypeptide (e.g., a single-chain polypeptide,antibody, antigen-binding fragment thereof, or construct thereof)further contains:

-   -   (a) a CDR-H2 having the amino acid sequence SSGZ⁴Z³Y (SEQ ID        NO: 263) or VDPEYZ⁴Z³T (SEQ ID NO: 264);    -   (b) a CDR-H3 having the amino acid sequence        QZ¹VZ²Z⁴YZ³SZ⁵WYZ⁵Z²Z⁵ (SEQ ID NO: 265) or        AZ¹DZ²Z⁴Z³Z⁵SPZ⁵Z²Z⁵WG (SEQ ID NO: 266);    -   (c) a CDR-L1 having the amino acid sequence SASSSVYYMZ⁵ (SEQ ID        NO: 267) or QNINKZ⁵ (SEQ ID NO: 268);        -   (d) a CDR-L2 having the amino acid sequence STSNLAZ³ (SEQ ID            NO: 269), TYZ³, or YTZ³; and/or        -   (e) a CDR-L3 having the amino acid sequence QQRRNZ⁵PYZ³ (SEQ            ID NO: 270) or CLQZ⁵VNLXZ³ (SEQ ID NO: 271);        -   in which each Z¹ is independently an amino acid including a            cationic side-chain at physiological pH;        -   each Z² is independently an amino acid including an anionic            side-chain at physiological pH;        -   each Z³ is independently an amino acid including a polar,            uncharged side-chain at physiological pH;        -   each Z⁴ is independently a glycine or alanine;        -   each Z⁵ is independently an amino acid including a            hydrophobic side-chain; and        -   each X is independently leucine or isoleucine.

The polypeptide (e.g., a single-chain polypeptide, antibody,antigen-binding fragment thereof, or construct thereof) may contain aCDR-H1 having the amino acid sequence GFTFSSY (SEQ ID NO: 23), GYTFTDYX(SEQ ID NO: 257), or an amino acid sequence having up to two amino acidsubstitutions (e.g., conservative amino acid substitutions) relative tothese sequences, in which each X is independently leucine or isoleucine,optionally in which the amino acid substitutions are conservative aminoacid substitutions. In some embodiments, the polypeptide (e.g., asingle-chain polypeptide, antibody, antigen-binding fragment thereof, orconstruct thereof) further contains:

-   -   (a) a CDR-H2 having the amino acid sequence SSGGSY (SEQ ID NO:        24), VDPEYGST (SEQ ID NO: 258), or an amino acid sequence having        up to two amino acid substitutions (e.g., conservative amino        acid substitutions) relative to these sequences;    -   (b) a CDR-H3 having the amino acid sequence QRVDGYSSYWYFDV (SEQ        ID NO: 25), ARDDGSYSPFDYWG (SEQ ID NO: 259), ARDDGSYSPFDY (SEQ        ID NO: 296), or an amino acid sequence having up to two amino        acid substitutions (e.g., conservative amino acid substitutions)        relative to these sequences;    -   (c) a CDR-L1 having the amino acid sequence SASSSVYYMY (SEQ ID        NO: 26), QNINKY (SEQ ID NO: 260), or an amino acid sequence        having up to two amino acid substitutions (e.g., conservative        amino acid substitutions) relative to these sequences;    -   (d) a CDR-L2 having the amino acid sequence STSNLAS (SEQ ID NO:        27), TYS, YTS, or an amino acid sequence having up to two amino        acid substitutions (e.g., conservative amino acid substitutions)        relative to SEQ ID NO: 27; and/or    -   (e) a CDR-L3 having the amino acid sequence QQRRNYPYT (SEQ ID        NO: 28), CLQYVNLXT (SEQ ID NO: 261), or an amino acid sequence        having up to two amino acid substitutions (e.g., conservative        amino acid substitutions) relative to these sequences.

In some embodiments, the polypeptide (e.g., a single-chain polypeptide,antibody, antigen-binding fragment thereof, or construct thereof)contains a heavy chain including one or more of the following CDRs:

-   -   (a) a CDR-H1 having the amino acid sequence GFTFSSY (SEQ ID NO:        23);    -   (b) a CDR-H2 having the amino acid sequence SSGGSY (SEQ ID NO:        24); and    -   (c) a CDR-H3 having the amino acid sequence QRVDGYSSYWYFDV (SEQ        ID NO: 25).

The polypeptide (e.g., a single-chain polypeptide, antibody,antigen-binding fragment thereof, or construct thereof) may contain, forexample, a heavy chain having one or more of the following CDRs:

-   -   (a) a CDR-H1 having the amino acid sequence GYTFTDYX (SEQ ID NO:        257);    -   (b) a CDR-H2 having the amino acid sequence VDPEYGST (SEQ ID NO:        258); and    -   (c) a CDR-H3 having the amino acid sequence ARDDGSYSPFDYWG (SEQ        ID NO: 259);    -   in which each X is independently leucine or isoleucine.

In some embodiments, the CDR-H1 has the amino acid sequence GYTFTDYL(SEQ ID NO: 274). In some embodiments, the CDR-H1 has the amino acidsequence GYTFTDYI (SEQ ID NO: 275). In some embodiments, the CDR-H1 hasthe amino acid sequence GYTFTDVI (SEQ ID NO: 293). In some embodiments,the CDR-H1 has the amino acid sequence GYTFTDYS (SEQ ID NO: 294).

Additionally or alternatively, the polypeptide (e.g., a single-chainpolypeptide, antibody, antigen-binding fragment thereof, or constructthereof) may contain, for example, a light chain having one or more ofthe following CDRs:

-   -   (a) a CDR-L1 having the amino acid sequence SASSSVYYMY (SEQ ID        NO: 26);    -   (b) a CDR-L2 having the amino acid sequence STSNLAS (SEQ ID NO:        27); and    -   (c) a CDR-L3 having the amino acid sequence QQRRNYPYT (SEQ ID        NO: 28).

In some embodiments, the antibody or antigen-binding fragment thereofcontains a light chain having one or more of the following CDRs:

-   -   (a) a CDR-L1 having the amino acid sequence QNINKY (SEQ ID NO:        260);    -   (b) a CDR-L2 having the amino acid sequence TYS or YTS; and    -   (c) a CDR-L3 having the amino acid sequence CLQYVNLXT (SEQ ID        NO: 261);    -   in which each X is independently leucine or isoleucine.

In some embodiments, the CDR-L2 has the amino acid sequence TYS. In someembodiments, the CDR-L2 has the amino acid sequence YTS. The CDR-L3 mayhave the amino acid sequence CLQYVNLLT (SEQ ID NO: 272). In someembodiments, the CDR-L3 has the amino acid sequence CLQYVNLIT (SEQ IDNO: 273).

The polypeptide (e.g., a single-chain polypeptide, antibody,antigen-binding fragment thereof, or construct thereof) may containthree heavy chain CDRs, including:

-   -   (a) a CDR-H1 having the amino acid sequence GFTFSSY (SEQ ID NO:        23);        -   (b) a CDR-H2 having the amino acid sequence SSGGSY (SEQ ID            NO: 24); and        -   (c) a CDR-H3 having the amino acid sequence QRVDGYSSYWYFDV            (SEQ ID NO: 25);        -   and may further contain three light chain CDRs, including:        -   (d) a CDR-L1 having the amino acid sequence SASSSVYYMY (SEQ            ID NO: 26);        -   (e) a CDR-L2 having the amino acid sequence STSNLAS (SEQ ID            NO: 27); and        -   (f) a CDR-L3 having the amino acid sequence QQRRNYPYT (SEQ            ID NO: 28).

In some embodiments, polypeptide (e.g., single-chain polypeptides,antibody, antigen-binding fragment thereof, or construct thereof)contains three heavy chain CDRs, including:

-   -   (a) a CDR-H1 having the amino acid sequence GYTFTDYX (SEQ ID NO:        257), such as GYTFTDYL (SEQ ID NO: 274) or GYTFTDYI (SEQ ID NO:        275);    -   (b) a CDR-H2 having the amino acid sequence VDPEYGST (SEQ ID NO:        258); and    -   (c) a CDR-H3 having the amino acid sequence ARDDGSYSPFDYWG (SEQ        ID NO: 259);    -   and further contains three light chain CDRs, including:    -   (d) a CDR-L1 having the amino acid sequence QNINKY (SEQ ID NO:        260);    -   (e) a CDR-L2 having the amino acid sequence TYS or YTS; and    -   (f) a CDR-L3 having the amino acid sequence CLQYVNLXT (SEQ ID        NO: 261), such as CLQYVNLLT (SEQ ID NO: 272) or CLQYVNLIT (SEQ        ID NO: 273);    -   in which each X is independently leucine or isoleucine.

In some embodiments, the polypeptide (e.g., a single-chain polypeptide,antibody, antigen-binding fragment thereof, or construct thereof)includes a framework region having the amino acid sequence LLIR (SEQ IDNO: 262) bound to the N-terminus of the CDR-L2 and/or a framework regionhaving the amino acid sequence TLE bound to the C-terminus of theCDR-L2.

The polypeptide (e.g., a single-chain polypeptide, antibody,antigen-binding fragment thereof, or construct thereof) may have a heavychain variable domain having an amino acid sequence that is at least 85%identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the aminoacid sequence of SEQ ID NO: 2. In some embodiments, the heavy chainvariable domain has an amino acid sequence that is at least 90%identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% identical) to the amino acid sequence of SEQ ID NO:2. In some embodiments, the heavy chain variable domain has an aminoacid sequence that is at least 95% identical (e.g., at least 95%, 96%,97%, 98%, 99%, 99.9%, or 100% identical) to the amino acid sequence ofSEQ ID NO: 2.

Additionally or alternatively, the polypeptide (e.g., a single-chainpolypeptide, antibody, antigen-binding fragment thereof, or constructthereof) may have a light chain variable domain having an amino acidsequence that is at least 85% identical (e.g., at least 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or100% identical) to the amino acid sequence of SEQ ID NO: 4. In someembodiments, the light chain variable domain has an amino acid sequencethat is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the amino acidsequence of SEQ ID NO: 4. In some embodiments, the light chain variabledomain has an amino acid sequence that is at least 95% identical (e.g.,at least 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical) to the aminoacid sequence of SEQ ID NO: 4.

In some embodiments, the polypeptide (e.g., a single-chain polypeptide,antibody, antigen-binding fragment thereof, or construct thereof)specifically binds to a peptide having the amino acid sequence of anyone of SEQ ID NOs: 11, 19, 20, and 34-117 with a K_(D) of less thanabout 100 nM (e.g., with a K_(D) of from about 10 pM to about 99 nM,such as a K_(D) of from about 20 pM to about 80 nM, from about 30 pM toabout 70 nM, from about 40 pM to about 60 nM, from about 50 pM to about50 nM, from about 60 pM to about 40 nM, from about 70 pM to about 30 nM,from about 80 pM to about 20 nM, from about 90 pM to about 10 nM, orfrom about 100 pM to about 1 nM) and does not specifically bind apeptide containing amino acids 56-60 (KCSPG) of SEQ ID NO: 7. Thepolypeptide (e.g., a single-chain polypeptide, antibody, antigen-bindingfragment thereof, or construct thereof) may bind the peptide having theamino acid sequence of any one of SEQ ID NOs: 11, 19, 20, and 34-117with a K_(D), e.g., of about 1 pM, 5 pM, 10 pM, 15 pM, 20 pM, 25 pM, 30pM, 35 pM, 40 pM, 45 pM, 50 pM, 55 pM, 60 pM, 65 pM, 70 pM, 75 pM, 80pM, 85 pM, 90 pM, 95 pM, 100 pM, 105 pM, 110 pM, 115 pM, 120 pM, 125 pM,130 pM, 135 pM, 140 pM, 145 pM, 150 pM, 155 pM, 160 pM, 165 pM, 170 pM,175 pM, 180 pM, 185 pM, 190 pM, 195 pM, 200 pM, 205 pM, 210 pM, 215 pM,220 pM, 225 pM, 230 pM, 235 pM, 240 pM, 245 pM, 250 pM, 255 pM, 260 pM,265 pM, 270 pM, 275 pM, 280 pM, 285 pM, 290 pM, 295 pM, 300 pM, 305 pM,310 pM, 315 pM, 320 pM, 325 pM, 330 pM, 335 pM, 340 pM, 345 pM, 350 pM,355 pM, 360 pM, 365 pM, 370 pM, 375 pM, 380 pM, 385 pM, 390 pM, 395 pM,400 pM, 405 pM, 410 pM, 415 pM, 420 pM, 425 pM, 430 pM, 435 pM, 440 pM,445 pM, 450 pM, 455 pM, 460 pM, 465 pM, 470 pM, 475 pM, 480 pM, 485 pM,490 pM, 495 pM, 500 pM, 505 pM, 510 pM, 515 pM, 520 pM, 525 pM, 530 pM,535 pM, 540 pM, 545 pM, 550 pM, 555 pM, 560 pM, 565 pM, 570 pM, 575 pM,580 pM, 585 pM, 590 pM, 595 pM, 600 pM, 605 pM, 610 pM, 615 pM, 620 pM,625 pM, 630 pM, 635 pM, 640 pM, 645 pM, 650 pM, 655 pM, 660 pM, 665 pM,670 pM, 675 pM, 680 pM, 685 pM, 690 pM, 695 pM, 700 pM, 705 pM, 710 pM,715 pM, 720 pM, 725 pM, 730 pM, 735 pM, 740 pM, 745 pM, 750 pM, 755 pM,760 pM, 765 pM, 770 pM, 775 pM, 780 pM, 785 pM, 790 pM, 795 pM, 800 pM,805 pM, 810 pM, 815 pM, 820 pM, 825 pM, 830 pM, 835 pM, 840 pM, 845 pM,850 pM, 855 pM, 860 pM, 865 pM, 870 pM, 875 pM, 880 pM, 885 pM, 890 pM,895 pM, 900 pM, 905 pM, 910 pM, 915 pM, 920 pM, 925 pM, 930 pM, 935 pM,940 pM, 945 pM, 950 pM, 955 pM, 960 pM, 965 pM, 970 pM, 975 pM, 980 pM,985 pM, 990 pM, 995 pM, 1 nM, 5 nM, 10 nM, 15 nM, 20 nM, 25 nM, 30 nM,35 nM, 40 nM, 45 nM, 50 nM, 55 nM, 60 nM, 65 nM, 70 nM, 75 nM, 80 nM, 85nM, 90 nM, 95 nM, 96 nM, 97 nM, 98 nM, or 99 nM, among other values.

The polypeptide (e.g., a single-chain polypeptide, antibody,antigen-binding fragment thereof, or construct thereof) may specificallybind TNFR2 at an epitope within:

-   -   (a) amino acids 142-146 of SEQ ID NO: 7 (KCRPG);    -   (b) amino acids 142-149 of SEQ ID NO: 7 (KCRPGFGV);    -   (c) amino acids 137-144 of SEQ ID NO: 7 (CAPLRKCR);    -   (d) amino acids 150-190 of SEQ ID NO: 7        (RPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAI);    -   (e) amino acids 161-169 of SEQ ID NO: 7 (CKPCAPGTF);    -   (f) amino acids 75-128 of SEQ ID NO: 7        (CDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCAL),        optionally in which the epitope is within amino acids 80-86        (DSTYTQL), 91-98 (PECLSCGS), or 116-123 (RICTCRPG) of SEQ ID NO:        7;    -   (g) amino acids 174-184 (SSTDICRPHQI) of SEQ ID NO: 7;    -   (h) amino acids 126-140 (CALSKQEGCRLCAPL) of SEQ ID NO: 7;        and/or    -   (i) amino acids 156-165 (TSDVVCKPCA) of SEQ ID NO: 7.

In some embodiments, the polypeptide (e.g., a single-chain polypeptide,antibody, antigen-binding fragment thereof, or construct thereof)specifically binds TNFR2 at two or more of the foregoing epitopes (e.g.,at two, three, four, five, six, seven, eight, nine, ten, or moreepitopes within the amino acid ranges set forth above).

In some embodiments, the polypeptide (e.g., a single-chain polypeptide,antibody, antigen-binding fragment thereof, or construct thereof)specifically binds TNFR2 with a K_(D) of no greater than about 10 nM,such as a K_(D) of no greater than about 1 nM. For example, thepolypeptide (e.g., a single-chain polypeptide, antibody, antigen-bindingfragment thereof, or construct thereof) may specifically bind TNFR2 witha K_(D) of from about 1 pM to about 10 nM, such as a K_(D) of about 1pM, 5 pM, 10 pM, 15 pM, 20 pM, 25 pM, 30 pM, 35 pM, 40 pM, 45 pM, 50 pM,55 pM, 60 pM, 65 pM, 70 pM, 75 pM, 80 pM, 85 pM, 90 pM, 95 pM, 100 pM,105 pM, 110 pM, 115 pM, 120 pM, 125 pM, 130 pM, 135 pM, 140 pM, 145 pM,150 pM, 155 pM, 160 pM, 165 pM, 170 pM, 175 pM, 180 pM, 185 pM, 190 pM,195 pM, 200 pM, 205 pM, 210 pM, 215 pM, 220 pM, 225 pM, 230 pM, 235 pM,240 pM, 245 pM, 250 pM, 255 pM, 260 pM, 265 pM, 270 pM, 275 pM, 280 pM,285 pM, 290 pM, 295 pM, 300 pM, 305 pM, 310 pM, 315 pM, 320 pM, 325 pM,330 pM, 335 pM, 340 pM, 345 pM, 350 pM, 355 pM, 360 pM, 365 pM, 370 pM,375 pM, 380 pM, 385 pM, 390 pM, 395 pM, 400 pM, 405 pM, 410 pM, 415 pM,420 pM, 425 pM, 430 pM, 435 pM, 440 pM, 445 pM, 450 pM, 455 pM, 460 pM,465 pM, 470 pM, 475 pM, 480 pM, 485 pM, 490 pM, 495 pM, 500 pM, 505 pM,510 pM, 515 pM, 520 pM, 525 pM, 530 pM, 535 pM, 540 pM, 545 pM, 550 pM,555 pM, 560 pM, 565 pM, 570 pM, 575 pM, 580 pM, 585 pM, 590 pM, 595 pM,600 pM, 605 pM, 610 pM, 615 pM, 620 pM, 625 pM, 630 pM, 635 pM, 640 pM,645 pM, 650 pM, 655 pM, 660 pM, 665 pM, 670 pM, 675 pM, 680 pM, 685 pM,690 pM, 695 pM, 700 pM, 705 pM, 710 pM, 715 pM, 720 pM, 725 pM, 730 pM,735 pM, 740 pM, 745 pM, 750 pM, 755 pM, 760 pM, 765 pM, 770 pM, 775 pM,780 pM, 785 pM, 790 pM, 795 pM, 800 pM, 805 pM, 810 pM, 815 pM, 820 pM,825 pM, 830 pM, 835 pM, 840 pM, 845 pM, 850 pM, 855 pM, 860 pM, 865 pM,870 pM, 875 pM, 880 pM, 885 pM, 890 pM, 895 pM, 900 pM, 905 pM, 910 pM,915 pM, 920 pM, 925 pM, 930 pM, 935 pM, 940 pM, 945 pM, 950 pM, 955 pM,960 pM, 965 pM, 970 pM, 975 pM, 980 pM, 985 pM, 990 pM, 995 pM, 1 nM, 5nM, or 10 nM, among other values. In some embodiments, the polypeptide(e.g., a single-chain polypeptide, antibody, antigen-binding fragmentthereof, or construct thereof) specifically binds TNFR2 with a K_(D) ofabout 621 pM. In some embodiments, the polypeptide (e.g., a single-chainpolypeptide, antibody, antigen-binding fragment thereof, or constructthereof) specifically binds TNFR2 with a K_(D) of about 44 pM.

The polypeptide (e.g., a single-chain polypeptide, antibody,antigen-binding fragment thereof, or construct thereof) may specificallybind TNFR2 to form an antibody-antigen complex with a k_(on) of at leastabout 10⁴ M⁻¹ s⁻¹, such as a k_(on) of from about 1×10⁴ M⁻¹ s⁻¹ to about1×10⁸ M⁻¹ s⁻¹. For example, the polypeptide (e.g., a single-chainpolypeptide, antibody, antigen-binding fragment thereof, or constructthereof) may specifically bind TNFR2 to form an antibody-antigen complexwith a k_(on) of about 1×10⁴ M⁻¹ s⁻¹, 2×10⁴ M⁻¹ s⁻¹, 3×10⁴ M⁻¹ s⁻¹,4×10⁴ M⁻¹ s⁻¹, 5×10⁴ M⁻¹ s⁻¹, 6×10⁴ M⁻¹ s⁻¹, 7×10⁴ M⁻¹ s⁻¹, 8×10⁴ M⁻¹s⁻¹, 9×10⁴ M⁻¹ s⁻¹, 1×10⁵ M⁻¹ s⁻¹, 2×10⁵ M⁻¹ s⁻¹, 3×10⁵ M⁻¹ s⁻¹, 4×10⁵M⁻¹ s⁻¹, 5×10⁵ M⁻¹ s⁻¹, 6×10⁵ M⁻¹ s⁻¹, 7×10⁵ M⁻¹ s⁻¹, 8×10⁵ M⁻¹ s⁻¹,9×10⁵ M⁻¹ s⁻¹, 1×10⁶ M⁻¹ s⁻¹, 2×10⁶ M⁻¹ s⁻¹, 3×10⁶ M⁻¹ s⁻¹, 4×10⁶ M⁻¹s⁻¹, 5×10⁶ M⁻¹ s⁻¹, 6×10⁶ M⁻¹ s⁻¹, 7×10⁶ M⁻¹ s⁻¹, 8×10⁶ M⁻¹ s⁻¹, 9×10⁶M⁻¹ s⁻¹, 1×10⁷ M⁻¹ s⁻¹, 2×10⁷ M⁻¹ s⁻¹, 3×10⁷ M⁻¹ s⁻¹, 4×10⁷ M⁻¹ s⁻¹,5×10⁷ M⁻¹ s⁻¹, 6×10⁷ M⁻¹ s⁻¹, 7×10⁷ M⁻¹ s⁻¹, 8×10⁷ M⁻¹ s⁻¹, 9×10⁷ M⁻¹s⁻¹, or 1×10⁸ M⁻¹ s⁻¹. In some embodiments, the polypeptide (e.g., asingle-chain polypeptide, antibody, antigen-binding fragment thereof, orconstruct thereof) specifically binds TNFR2 to form an antibody-antigencomplex with a k_(on) of about 4.9×10⁶ M⁻¹ s⁻¹. In some embodiments, thepolypeptide (e.g., a single-chain polypeptide, antibody, antigen-bindingfragment thereof, or construct thereof) specifically binds TNFR2 to forman antibody-antigen complex with a k_(on) of about 3.6×10⁵ M⁻¹ s⁻¹.

The polypeptide (e.g., a single-chain polypeptide, antibody,antigen-binding fragment thereof, or construct thereof) may specificallybind TNFR2 to form an antibody-antigen complex that dissociates with ak_(off) of, for example, no greater than about 10⁻³ s⁻¹, such as ak_(off) of from about 10⁻⁶ s⁻¹ to about 10⁻³ s⁻¹ (e.g., a k_(off) ofabout 1×10⁻⁶ s⁻¹, 2×10⁻⁶ s⁻¹, 3×10⁻⁶ s⁻¹, 4×10⁻⁶ s⁻¹, 5×10⁻⁶ s⁻¹, 6×10⁻⁶s⁻¹, 7×10⁻⁶ s⁻¹, 8×10⁻⁶ s⁻¹, 9×10⁻⁶ s⁻¹, 1×10⁻⁵ s⁻¹, 2×10⁻⁵ s⁻¹, 3×10⁻⁵s⁻¹, 4×10⁻⁵ s⁻¹, 5×10⁻⁵ s⁻¹, 6×10⁻⁵ s⁻¹, 7×10⁻⁵ s⁻¹, 8×10⁻⁵ s⁻¹, 9×10⁻⁵s⁻¹, 1×10⁻⁴ s⁻¹, 2×10⁻⁴ s⁻¹, 3×10⁻⁴ s⁻¹, 4×10⁻⁴ s⁻¹, 5×10⁻⁴ s⁻¹, 6×10⁻⁴s⁻¹, 7×10⁻⁴ s⁻¹, 8×10⁻⁴ s⁻¹, 9×10⁻⁴ s⁻¹, or 1×10⁻³ s⁻¹. In someembodiments, the antibody-antigen complex dissociates with a k_(off) ofabout 2.2×10⁻⁴ s⁻¹.

Polypeptides, such as single-chain polypeptides, antibodies,antigen-binding fragments thereof, and constructs thereof, describedherein may inhibit TNFR2 signaling, e.g., in a cell that expressesTNFR2, such as a T-reg cell (e.g., a T-reg cell that expressesCD25^(Hi)), myeloid-derived suppressor cell (MDSC), and/or a TNFR2+cancer cell. In some embodiments, the single-chain polypeptide,antibody, or antigen-binding fragment thereof reduces or inhibits theexpression of one or more genes selected from the group consisting ofCHUK, NFKBIE, NFKBIA, MAP3K11, TRAF2, TRAF3, relB, and cIAP2/BIRC3, asassessed, for example, by observing a decrease in the expression of oneor more of the above genes or by other methods known in the art forassessing gene activation. For instance, antagonistic TNFR2 single-chainpolypeptides, antibodies, antigen-binding fragments thereof, andconstructs thereof may inhibit the expression or post-translationalmodification (e.g., phosphorylation) of one or more of CHUK, NFKBIE,NFKBIA, MAP3K11, TRAF2, TRAF3, relB, or cIAP2/BIRC3, e.g., by about 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative tothe expression or post-translational modification (e.g.,phosphorylation) of one or more of these proteins isolated from a samplenot treated with an antagonistic TNFR2 single-chain polypeptide,antibody, antigen-binding fragment thereof, or construct thereofdescribed herein. Exemplary assays that can be used to determineexpression level and phosphorylation state are known in the art andinclude, e.g., Western blot assays to determine protein content andquantitative reverse transcription polymerase chain reaction (RT-PCR)experiments to determine mRNA content. In preferred embodiments,anti-TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies,antigen-binding fragments thereof, and constructs thereof) are dominantTNFR2 antagonists and are, thus, capable of inhibiting TNFR2 activationeven in the presence of a TNFR2 agonist (such as, e.g., TNFα or BacillusCalmette-Guérin (BCG)) or a growth-promoting agent, such as IL-2.

Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides,antibodies, antigen-binding fragments thereof, and constructs thereof)described herein may exhibit one or more, or all, of the followingproperties:

-   -   (a) Suppression of the proliferation of, and/or direct killing        of, T-reg cells (e.g., thereby reducing the quantity of T-reg        cells in a population of cells by about 1%, 2%, 3%, 4%, 5%, 6%,        7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,        60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%,        99.9%, or 100%, relative to a population of cells not exposed to        the polypeptide), for instance, by binding and inactivating        TNFR2 on the T-reg cell surface;    -   (b) Suppression of the proliferation of, and/or direct killing        of, MDSCs (e.g., thereby reducing the quantity of MDSCs in a        population of cells by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,        10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,        75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100%,        relative to a population of cells not exposed to the        polypeptide), for instance, by binding and inactivating TNFR2 on        the MDSC surface;    -   (c) Promotion of the expansion of T effector cells, such as CD8+        T cells (e.g., thereby increasing the quantity of CD8+ effector        T cells in a population of cells by about 1.1-fold, 1.2-fold,        1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold,        1.9-fold, 2-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold,        2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3-fold,        3.1-fold, 3.2-fold, 3.3-fold, 3.4-fold, 3.5-fold, 3.6-fold,        3.7-fold, 3.8-fold, 3.9-fold, 4-fold, 4.1-fold, 4.2-fold,        4.3-fold, 4.4-fold, 4.5-fold, 4.6-fold, 4.7-fold, 4.8-fold,        4.9-fold, 5-fold, 5.1-fold, 5.2-fold, 5.3-fold, 5.4-fold,        5.5-fold, 5.6-fold, 5.7-fold, 5.8-fold, 5.9-fold, 6-fold,        6.1-fold, 6.2-fold, 6.3-fold, 6.4-fold, 6.5-fold, 6.6-fold,        6.7-fold, 6.8-fold, 6.9-fold, 7-fold, 7.1-fold, 7.2-fold,        7.3-fold, 7.4-fold, 7.5-fold, 7.6-fold, 7.7-fold, 7.8-fold,        7.9-fold, 8-fold, 8.1-fold, 8.2-fold, 8.3-fold, 8.4-fold,        8.5-fold, 8.6-fold, 8.7-fold, 8.8-fold, 8.9-fold, 9-fold,        9.1-fold, 9.2-fold, 9.3-fold, 9.4-fold, 9.5-fold, 9.6-fold,        9.7-fold, 9.8-fold, 9.9-fold, 10-fold, 20-fold, 30-fold,        40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold,        or more, relative to a population of cells not exposed to the        polypeptide); and/or    -   (d) Suppression of the proliferation of, and/or direct killing        of, TNFR2-expressing cancer cells, such as a Hodgkin's lymphoma        cell, a cutaneous non-Hodgkin's lymphoma cell, a T cell lymphoma        cell, an ovarian cancer cell, a colon cancer cell, a multiple        myeloma cell, a renal cell carcinoma cell, a skin cancer cell, a        lung cancer cell, a liver cancer cell, an endometrial cancer        cell, a hematopoietic or lymphoid cancer cell, a central nervous        system cancer cell, a breast cancer cell, a pancreatic cancer        cell, a stomach cancer cell, an esophageal cancer cell, and an        upper gastrointestinal cancer cell (e.g., thereby reducing the        quantity of TNFR2-expressing cancer cells in a population of        cells by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%,        20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,        85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100%, relative to a        population of cells not exposed to the polypeptide).

For example, an antagonistic TNFR2 polypeptide, such as a single-chainpolypeptide, antibody, antigen-binding fragment thereof, and constructthereof, described herein can be used to reduce the total quantity ofT-reg or cancer cells in a patient (such as a human patient) or within asample (e.g., a sample isolated from a patient, such as a human patientundergoing treatment for cancer or an infectious disease as describedherein) relative to a patient or sample, respectively, not treated withthe polypeptide.

In some embodiments, the antagonistic TNFR2 polypeptide (e.g., asingle-chain polypeptide, antibody, or antigen-binding fragment thereof)reduces expression of TNFR2, e.g., by a T-reg cell or a cancer cell(such as a TNFR2+ cancer cell, e.g., a Hodgkin's lymphoma cell, acutaneous non-Hodgkin's lymphoma cell, a T cell lymphoma cell, anovarian cancer cell, a colon cancer cell, a multiple myeloma cell, arenal cell carcinoma cell, a skin cancer cell, a lung cancer cell, aliver cancer cell, an endometrial cancer cell, a hematopoietic orlymphoid cancer cell, a central nervous system cancer cell, a breastcancer cell, a pancreatic cancer cell, a stomach cancer cell, anesophageal cancer cell, or an upper gastrointestinal cancer cell),and/or the secretion of soluble TNFR2 by one or more of the foregoingcells.

An antagonistic TNFR2 polypeptide (e.g., a single-chain polypeptide,antibody, antigen-binding fragment thereof, and construct thereof)described herein can be used to inhibit or reduce the proliferation of,or reduce the total quantity of, T-reg cells in a patient (e.g., a humanpatient) or in a sample (e.g., a sample isolated from a human patientundergoing treatment for cancer or an infectious disease as describedherein).

An antagonistic TNFR2 polypeptide (e.g., a single-chain polypeptide,antibody, antigen-binding fragment thereof, and construct thereof)described herein can be used to inhibit or reduce the proliferation of,and/or to directly kill, T-reg cells (e.g., activated T-reg cells thatexpress CD25^(Hi)) and/or cancer cells that express TNFR2. For instance,the cancer cells may be selected from the group consisting of aHodgkin's lymphoma cell, a cutaneous non-Hodgkin's lymphoma cell, a Tcell lymphoma cell, an ovarian cancer cell, a colon cancer cell, amultiple myeloma cell, a renal cell carcinoma cell, a skin cancer cell,a lung cancer cell, a liver cancer cell, an endometrial cancer cell, ahematopoietic or lymphoid cancer cell, a central nervous system cancercell, a breast cancer cell, a pancreatic cancer cell, a stomach cancercell, an esophageal cancer cell, or an upper gastrointestinal cancercell. Without being limited by mechanism, binding of TNFR2 on the cancercell may inhibit or reduce proliferation of the cancer cell and/or maydirectly kill the cancer cell, such as by promoting apoptosis of thecancer cell.

Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides,antibodies, antigen-binding fragments thereof, and constructs thereof)described herein bind TNFR2 on the surface of a MDSC (e.g., a cell thatexpresses all or a subset of proteins and small molecules selected fromthe group consisting of B7-1 (CD80), B7-H1 (PD-L1), CCR2, CD1d, CD1d1,CD2, CD31 (PECAM-1), CD43, CD44, complement component C5a R1, F4/80(EMR1), Fcγ RIII (CD16), Fcγ RII (CD32), Fcγ RIIA (CD32a), Fcγ RIIB(CD32b), Fcγ RIIB/C (CD32b/c), Fcγ RIIC (CD32c), Fcγ RIIIA (CD16A), FcγRIIIB (CD16b), galectin-3, GP130, Gr-1 (Ly-6G), ICAM-1 (CD54), IL-1 RI,IL-4Rα, IL-6Rα, integrin α4 (CD49d), integrin αL (CD11a), integrin αM(CD11 b), M-CSFR, MGL1 (CD301a), MGL1/2 (CD301a/b), MGL2 (CD301b),nitric oxide, PSGL-1 (CD162), L-selectin (CD62L), siglec-3 (CD33),transferrin receptor (TfR), VEGFR1 (Flt-1), and VEGFR2 (KDR or Flk-1)).Particularly, MDSCs do not express proteins selected from the groupconsisting of B7-2 (CD86), B7-H4, CD11c, CD14, CD21, CD23 (FccRII),CD34, CD35, CD40 (TNFRSF5), CD117 (c-kit), HLA-DR, and Sca-1 (Ly6).Binding of TNFR2 on the MDSC may inhibit or reduce proliferation of theMDSC and/or may directly kill the MDSC, such as by promoting apoptosisof the MDSC. Polypeptides, such as single-chain polypeptides,antibodies, antigen-binding fragments thereof, and constructs thereof,described herein may not require TNFα to inhibit the proliferation ofT-reg cells, cancer cells (e.g., TNFR2-expressing cancer cells), and/orMDSCs.

In some embodiments, the polypeptides described herein, such assingle-chain polypeptides, antibodies, antigen-binding fragmentsthereof, and constructs thereof, inhibit the proliferation of, and/ordirectly kill, T-reg cells with a greater potency in a patient sufferingfrom cancer relative to a subject that does not have cancer. In someembodiments, the polypeptides described herein, such as single-chainpolypeptides, antibodies, antigen-binding fragments thereof, andconstructs thereof, inhibit the proliferation of, and/or directly kill,T-reg cells with a greater potency in the microenvironment of a tumorrelative to a site that is free of cancer cells, such as a site distalfrom a tumor in a patient suffering from cancer.

For example, in some embodiments, the polypeptides described herein,such as single-chain polypeptides, antibodies, antigen-binding fragmentsthereof, and constructs thereof, inhibit the proliferation of, and/ordirectly kill, T-reg cells with a potency that is greater in themicroenvironment of a tumor than in a site that is free of cancer cells,such as a site distal from a tumor in a patient suffering from cancer,or relative to a subject without cancer. For instance, the polypeptidesdescribed herein, such as single-chain polypeptides, antibodies,antigen-binding fragments thereof, and constructs thereof, may exhibitan IC₅₀ for inhibiting the proliferation of T-reg cells in a tumormicroenvironment that is less than the IC₅₀ of the polypeptides forinhibiting the proliferation of T-reg cells in a site that is free ofcancer cells by, for example, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold,1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold,4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold,20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 100-fold,1,000-fold, 10,000-fold, or more. The polypeptides described herein,such as single-chain polypeptides, antibodies, antigen-binding fragmentsthereof, and constructs thereof, may inhibit the proliferation of T-regcells with a potency that is greater in the microenvironment of a tumorcontaining T cell lymphoma cells (e.g., Hodgkin's or cutaneousnon-Hodgkin's lymphoma cells), ovarian cancer cells, colon cancer cells,multiple myeloma cells, or renal cell carcinoma cells than in a sitethat is free of such cancer cells, such as a site distal from a tumor ina patient suffering from one or more of the foregoing cancers, orrelative to a subject without cancer.

In some embodiments, the polypeptides described herein, such assingle-chain polypeptides, antibodies, antigen-binding fragmentsthereof, and constructs thereof, inhibit or reduce the proliferation of,and/or directly kill, MDSCs with a greater potency in a patientsuffering from cancer relative to a subject that does not have cancer.In some embodiments, the polypeptides described herein, such assingle-chain polypeptides, antibodies, antigen-binding fragmentsthereof, and constructs thereof, inhibit or reduce the proliferation of,and/or directly kill, MDSCs with a greater potency in themicroenvironment of a tumor relative to a site that is free of cancercells, such as a site distal from a tumor in a patient suffering fromcancer, or relative to a subject without cancer.

For example, antagonistic TNFR2 polypeptides (e.g., single-chainpolypeptides, antibodies, antigen-binding fragments thereof, andconstructs thereof) described herein may bind TNFR2 on the surface of aMDSC present within the microenvironment of a tumor, and may inhibit orreduce proliferation of the MDSC or may promote the apoptosis of theMDSC with a potency that is greater in the microenvironment of a tumorthan at a site that is free of cancer cells, such as a site distal froma tumor in a patient suffering from cancer, or relative to a subjectwithout cancer. For instance, the polypeptides described herein, such assingle-chain polypeptides, antibodies, antigen-binding fragmentsthereof, and constructs thereof, may exhibit an IC₅₀ for inhibiting theproliferation of MDSCs in a tumor microenvironment that is less than theIC₅₀ of the polypeptides for inhibiting the proliferation of MDSCs in asite that is free of cancer cells by, for example, 1.1-fold, 1.2-fold,1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold,2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold,100-fold, 1,000-fold, 10,000-fold, or more. The polypeptides describedherein, such as single-chain polypeptides, antibodies, antigen-bindingfragments thereof, and constructs thereof, may inhibit the proliferationof MDSCs or may promote the apoptosis of MDSCs with a potency that isgreater in the microenvironment of a tumor containing T cell lymphomacells (e.g., Hodgkin's or cutaneous non-Hodgkin's lymphoma cells),ovarian cancer cells, colon cancer cells, multiple myeloma cells, orrenal cell carcinoma cells than in a site that is free of such cancercells, such as a site distal from a tumor in a patient suffering fromone or more of the foregoing cancers, or relative to a subject withoutcancer.

In some embodiments, the polypeptides described herein, such assingle-chain polypeptides, antibodies, antigen-binding fragmentsthereof, and constructs thereof, expand T effector cells, such as CD8+cytotoxic T cells, with a greater potency in a patient suffering fromcancer relative to a subject that does not have cancer. In someembodiments, the polypeptides described herein, such as single-chainpolypeptides, antibodies, antigen-binding fragments thereof, andconstructs thereof, expand T effector cells, such as CD8+ cytotoxic Tcells, with a greater potency in the microenvironment of a tumorrelative to a site that is free of cancer cells, such as a site distalfrom a tumor in a patient suffering from cancer, or relative to asubject without cancer.

For instance, in some embodiments, the polypeptides described herein,such as single-chain polypeptides, antibodies, antigen-binding fragmentsthereof, and constructs thereof, directly expand T effector cells, suchas CD8+ cytotoxic T cells, with a potency that is greater in themicroenvironment of a tumor than in a site that is free of cancer cells,such as a site distal from a tumor in a patient suffering from cancer,or relative to a subject without cancer. For instance, the polypeptidesdescribed herein may have an EC₅₀ for expanding T effector cells in acancer patient that is less than the EC₅₀ of the polypeptides forexpanding T effector cells in a subject without cancer by, for example,1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold,1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold,40-fold, 45-fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold, or more.The polypeptides described herein, such as single-chain polypeptides,antibodies, antigen-binding fragments thereof, and constructs thereof,may directly expand T effector cells, such as CD8+ cytotoxic T cells,with a potency that is greater in the microenvironment of a tumorcontaining T cell lymphoma cells (e.g., Hodgkin's or cutaneousnon-Hodgkin's lymphoma cells), ovarian cancer cells, colon cancer cells,multiple myeloma cells, or renal cell carcinoma cells than in a sitethat is free of such cancer cells, such as a site distal from a tumor ina patient suffering from one or more of the foregoing cancers or in asubject without cancer. In some embodiments, the T effector cells (e.g.,CD8+ cytotoxic T cells) specifically react with an antigen present onone or more cancer cells, such as Hodgkin's lymphoma cells, cutaneousnon-Hodgkin's lymphoma cells, T cell lymphoma cells, ovarian cancercells, colon cancer cells, multiple myeloma cells, or renal cellcarcinoma cells.

In some embodiments, the polypeptide is a human IgG2 isotype antibody orantigen-binding fragment thereof. Additionally or alternatively, thepolypeptide may be an antibody or antigen-binding fragment thereofselected from the group consisting of a monoclonal antibody orantigen-binding fragment thereof, a polyclonal antibody orantigen-binding fragment thereof, a human antibody or antigen-bindingfragment thereof, a humanized antibody or antigen-binding fragmentthereof, a primatized antibody or antigen-binding fragment thereof, abispecific antibody or antigen-binding fragment thereof, amulti-specific antibody or antigen-binding fragment thereof, adual-variable immunoglobulin domain, a monovalent antibody orantigen-binding fragment thereof, a chimeric antibody or antigen-bindingfragment thereof, a single-chain Fv molecule (scFv), a diabody, atriabody, a nanobody, an antibody-like protein scaffold, a domainantibody, a Fv fragment, a Fab fragment, a F(ab′)₂ molecule, and atandem scFv (taFv). In some embodiments, the antibody or antigen-bindingfragment thereof contains two or more CDRs covalently bound to oneanother, e.g., by an amide bond, a thioether bond, a carbon-carbon bond,or a disulfide bridge, or by a linker, such as a linker describedherein. In some embodiments, the antibody or antigen-binding fragmentthereof is a human, humanized, or chimeric antibody or antigen-bindingfragment thereof.

In some embodiments, the polypeptide (e.g., a single-chain polypeptide,antibody, antigen-binding fragment thereof, or construct thereof) isconjugated to a therapeutic agent, such as a cytotoxic agent (e.g., acytotoxic agent described herein).

The antagonistic TNFR2 antibody of any of the above aspects can be abispecific antibody, such as a bispecific monoclonal antibody, in whichone arm of the antibody specifically binds TNFR2 and the otherspecifically binds an immune checkpoint protein, such as PD-1, PD-L1, orCTLA-4, among others described herein. The arm of the bispecificantibody that specifically binds TNFR2 may specifically bind, forexample, an epitope of human TNFR2 defined by one or more amino acidswithin CRD3 and/or an epitope defined by one or more amino acids withinCRD4. In some embodiments, the arm of the bispecific antibody thatspecifically binds TNFR2 specifically binds an epitope of human TNFR2selected from:

-   -   (a) amino acids 142-146 of SEQ ID NO: 7 (KCRPG);    -   (b) amino acids 142-149 of SEQ ID NO: 7 (KCRPGFGV);    -   (c) amino acids 137-144 of SEQ ID NO: 7 (CAPLRKCR);    -   (d) amino acids 150-190 of SEQ ID NO: 7        (RPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAI);        -   (e) amino acids 161-169 of SEQ ID NO: 7 (CKPCAPGTF);        -   (f) amino acids 75-128 of SEQ ID NO: 7            (CDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCAL),            optionally in which the epitope is within amino acids 80-86            (DSTYTQL), 91-98 (PECLSCGS), or 116-123 (RICTCRPG) of SEQ ID            NO: 7;        -   (g) amino acids 174-184 (SSTDICRPHQI) of SEQ ID NO: 7;        -   (h) amino acids 126-140 (CALSKQEGCRLCAPL) of SEQ ID NO: 7;            and        -   (i) amino acids 156-165 (TSDVVCKPCA) of SEQ ID NO: 7.

In some embodiments, the bispecific antibody contains one arm thatspecifically binds TNFR2, such as an epitope of human TNFR2 describedabove, and one arm that specifically binds an immune checkpoint proteinspecifically binds PD-1. In some embodiments, the arm of the bispecificantibody that specifically binds PD-1 may specifically bind the sameepitope(s) on PD-1 as nivolumab, pembrolizumab, avelumab, durvalumab, oratezolizumab. For example, the arm of the bispecific antibody thatspecifically binds PD-1 may competitively inhibit the binding of PD-1 tonivolumab, pembrolizumab, avelumab, durvalumab, and/or atezolizumab, asassessed, for example, using a competitive binding assay describedherein or know in the art, such as a competitive ELISA.

In some embodiments, the bispecific antibody contains one arm thatspecifically binds TNFR2, such as an epitope of human TNFR2 describedabove, and one arm that specifically binds PD-L1. In some embodiments,the arm of the bispecific antibody that specifically binds PD-L1 mayspecifically bind the same epitope(s) on PD-L1 as atezolizumab oravelumab. For example, the arm of the bispecific antibody thatspecifically binds PD-L1 may competitively inhibit the binding of PD-L1to atezolizumab and/or avelumab, for example, using a competitivebinding assay described herein or know in the art, such as a competitiveELISA.

In some embodiments, the bispecific antibody contains one arm thatspecifically binds TNFR2, such as an epitope of human TNFR2 describedabove, and one arm that specifically binds CTLA-4. In some embodiments,the arm of the bispecific antibody that specifically binds CTLA-4 mayspecifically bind the same epitope(s) on CTLA-4 as ipilimumab ortremelimumab. For example, the arm of the bispecific antibody thatspecifically binds CTLA-4 may competitively inhibit the binding ofCTLA-4 to ipilimumab and/or tremelimumab, as assessed, for example,using a competitive binding assay described herein or know in the art,such as a competitive ELISA.

A second aspect features a construct containing a first polypeptidedomain and a second polypeptide domain. The first polypeptide domain andthe second polypeptide domain are each, independently, anantigen-binding fragment of the first aspect or any of the embodimentsthereof. The first polypeptide domain and the second polypeptide domainmay be bound to one another, for example, by a covalent linker, such asa linker that contains (e.g., is) an amide bond or a disulfide bond.

A third aspect features a polynucleotide encoding the polypeptide (e.g.,a single-chain polypeptide, antibody, antigen-binding fragment thereof,or construct thereof) of the first aspect and/or the construct of thesecond aspect or any of the embodiments thereof.

A fourth aspect features a vector encoding the polynucleotide of thethird aspect. The vector may be an expression vector, such as aeukaryotic expression vector. In some embodiments, the vector is a viralvector, such as an adenovirus (e.g., a serotype 1-57 adenovirus, such asa serotype 2, 5, 11, 12, 24, 26, 34, 35, 40, 48, 49, 50, 52, or Pan9adenovirus), retrovirus (e.g., a γ-retrovirus or a lentivirus),poxvirus, adeno-associated virus, baculovirus, herpes simplex virus, ora vaccinia virus (e.g., a modified vaccinia Ankara virus).

A fifth aspect features an isolated host cell containing thepolynucleotide of the third aspect and/or the vector of the fourthaspect. The host cell may be a prokaryotic cell or a eukaryotic cell,such as a mammalian cell (e.g., a Chinese hamster ovary (CHO) cell). Thehost cell may be one that is described, e.g., in Dinnis and James,Biotechnology and Bioengineering 91:180-189, 2005, the disclosure ofwhich is incorporated herein by reference.

A sixth aspect features a pharmaceutical composition containing apolypeptide (e.g., a single-chain polypeptide, antibody, antigen-bindingfragment thereof, or construct thereof) that specifically binds humanTNFR2 and exhibits an antagonistic effect on TNFR2 activity upon thebinding. The polypeptide may be, for example, an antibody orantigen-binding fragment of the first aspect or any of the embodimentsthereof. Additionally or alternatively, the antibody or antigen-bindingfragment thereof may be one that specifically binds human TNFR2 at anepitope within CRD3 and/or CRD4 and does not bind TNFR2 at an epitopedefined by one or more amino acids within CRD1, in which at least 10% ofthe antibody or antigen-binding fragment thereof in the pharmaceuticalcomposition is present in a single disulfide-bonded isoform, such as theIgG2-A or IgG2-B disulfide-bonded isoform. In some embodiments, about10% to about 99.999% of the antibody or antigen-binding fragment thereofin the pharmaceutical composition is present in a singledisulfide-bonded isoform, such as from about 11% to about 99.9%, about12% to about 99.9%, about 13% to about 99.9%, about 14% to about 99.9%,about 15% to about 99%, about 16% to about 99.9%, about 17% to about99.9%, about 18% to about 99.9%, about 19% to about 99.9%, about 20% toabout 99.9%, about 21% to about 99.9%, about 22% to about 99.9%, about23% to about 99.9%, about 24% to about 99.9%, about 25% to about 99.9%,about 26% to about 99.9%, about 27% to about 99.9%, about 28% to about99.9%, about 29% to about 99.9%, about 30% to about 99.9%, about 31% toabout 99.9%, about 32% to about 99.9%, about 33% to about 99.9%, about34% to about 99.9%, about 35% to about 99.9%, about 36% to about 99.9%,about 37% to about 99.9%, about 38% to about 99.9%, about 39% to about99.9%, about 40% to about 99.9%, about 41% to about 99.9%, about 42% toabout 99.9%, about 43% to about 99.9%, about 44% to about 99.9%, about45% to about 99.9%, about 46% to about 99.9%, about 47% to about 99.9%,about 48% to about 99.9%, about 49% to about 99.9%, about 50% to about99.9%, about 51% to about 99.9%, about 52% to about 99.9%, about 53% toabout 99.9%, about 54% to about 99.9%, about 55% to about 99.9%, about56% to about 99.9%, about 57% to about 99.9%, about 58% to about 99.9%,about 59% to about 99.9%, about 60% to about 99.9%, about 61% to about99.9%, about 62% to about 99.9%, about 63% to about 99.9%, about 64% toabout 99.9%, about 65% to about 99.9%, about 66% to about 99.9%, about67% to about 99.9%, about 68% to about 99.9%, about 69% to about 99.9%,about 70% to about 99.9%, about 71% to about 99.9%, about 72% to about99.9%, about 73% to about 99.9%, about 74% to about 99.9%, about 75% toabout 99.9%, about 76% to about 99.9%, about 77% to about 99.9%, about78% to about 99.9%, about 79% to about 99.9%, about 80% to about 99.9%,about 81% to about 99.9%, about 82% to about 99.9%, about 83% to about99.9%, about 84% to about 99.9%, about 85% to about 99.9%, about 86% toabout 99.9%, about 87% to about 99.9%, about 88% to about 99.9%, about89% to about 99.9%, about 90% to about 99.9%, about 91% to about 99.9%,about 92% to about 99.9%, about 93% to about 99.9%, about 94% to about99.9%, about 95% to about 99.9%, about 96% to about 99.9%, about 97% toabout 99.9%, about 98% to about 99.9%, or about 99% to about 99.99% ofthe antibody or antigen-binding fragment thereof.

In some embodiments, at least about 10% of the antibody orantigen-binding fragment thereof in the pharmaceutical composition ispresent in a single disulfide-bonded isoform. In some embodiments, atleast about 15% of the antibody or antigen-binding fragment thereof inthe pharmaceutical composition is present in a single disulfide-bondedisoform. In some embodiments, at least about 20% of the antibody orantigen-binding fragment thereof in the pharmaceutical composition ispresent in a single disulfide-bonded isoform. In some embodiments, atleast about 25% of the antibody or antigen-binding fragment thereof inthe pharmaceutical composition is present in a single disulfide-bondedisoform. In some embodiments, at least about 30% of the antibody orantigen-binding fragment thereof in the pharmaceutical composition ispresent in a single disulfide-bonded isoform. In some embodiments, atleast about 35% of the antibody or antigen-binding fragment thereof inthe pharmaceutical composition is present in a single disulfide-bondedisoform. In some embodiments, at least about 40% of the antibody orantigen-binding fragment thereof in the pharmaceutical composition ispresent in a single disulfide-bonded isoform. In some embodiments, atleast about 45% of the antibody or antigen-binding fragment thereof inthe pharmaceutical composition is present in a single disulfide-bondedisoform. In some embodiments, at least about 50% of the antibody orantigen-binding fragment thereof in the pharmaceutical composition ispresent in a single disulfide-bonded isoform. In some embodiments, atleast about 60% of the antibody or antigen-binding fragment thereof inthe pharmaceutical composition is present in a single disulfide-bondedisoform. In some embodiments, at least about 65% of the antibody orantigen-binding fragment thereof in the pharmaceutical composition ispresent in a single disulfide-bonded isoform. In some embodiments, atleast about 70% of the antibody or antigen-binding fragment thereof inthe pharmaceutical composition is present in a single disulfide-bondedisoform. In some embodiments, at least about 75% of the antibody orantigen-binding fragment thereof in the pharmaceutical composition ispresent in a single disulfide-bonded isoform. In some embodiments, atleast about 80% of the antibody or antigen-binding fragment thereof inthe pharmaceutical composition is present in a single disulfide-bondedisoform. In some embodiments, at least about 85% of the antibody orantigen-binding fragment thereof in the pharmaceutical composition ispresent in a single disulfide-bonded isoform. In some embodiments, atleast about 90% of the antibody or antigen-binding fragment thereof inthe pharmaceutical composition is present in a single disulfide-bondedisoform. In some embodiments, at least about 95% of the antibody orantigen-binding fragment thereof in the pharmaceutical composition ispresent in a single disulfide-bonded isoform. In some embodiments, atleast about 96% of the antibody or antigen-binding fragment thereof inthe pharmaceutical composition is present in a single disulfide-bondedisoform. In some embodiments, at least about 97% of the antibody orantigen-binding fragment thereof in the pharmaceutical composition ispresent in a single disulfide-bonded isoform. In some embodiments, atleast about 98% of the antibody or antigen-binding fragment thereof inthe pharmaceutical composition is present in a single disulfide-bondedisoform. In some embodiments, at least about 99% of the antibody orantigen-binding fragment thereof in the pharmaceutical composition ispresent in a single disulfide-bonded isoform. In some embodiments, atleast about 99.9% of the antibody or antigen-binding fragment thereof inthe pharmaceutical composition is present in a single disulfide-bondedisoform.

In some embodiments, the antibody or antigen-binding fragment thereofyields only a single detectable band upon gel electrophoresis analysisperformed under non-reducing conditions.

In some embodiments, the single disulfide-bonded isoform of the antibodyor antigen-binding fragment is IgG2-A, as described herein. In someembodiments, the single disulfide-bonded isoform of the antibody orantigen-binding fragment is IgG2-B, as described herein.

Additionally or alternatively, the pharmaceutical composition maycontain the construct of the second aspect or any embodiments thereof,the polynucleotide of the third aspect or any embodiments thereof, thevector of the fourth aspect or any embodiments thereof, and/or the hostcell of the fifth aspect or any embodiments thereof. The pharmaceuticalcomposition may further contain a pharmaceutically acceptable carrier orexcipient.

In some embodiments, the polypeptide (e.g., a single-chain polypeptide,antibody, antigen-binding fragment thereof, or construct thereof) ispresent in the pharmaceutical composition in an amount of from about0.001 mg/ml to about 100 mg/ml, such as an amount of from about 0.01mg/ml to about 10 mg/ml.

The pharmaceutical composition may further contain an additionaltherapeutic agent, such as an immunotherapy agent. In some embodiments,the immunotherapy agent is selected from the group consisting of ananti-CTLA-4 agent, an anti-PD-1 agent, an anti-PD-L1 agent, ananti-PD-L2 agent, a TNF-α cross-linking agent, a TRAIL cross-linkingagent, an anti-CD27 agent, an anti-CD30 agent, an anti-CD40 agent, ananti-4-1 BB agent, an anti-GITR agent, an anti-OX40 agent, ananti-TRAILR1 agent, an anti-TRAILR2 agent, an anti-TWEAK agent, ananti-TWEAKR agent, an anti-cell surface lymphocyte protein agent, ananti-BRAF agent, an anti-MEK agent, an anti-CD33 agent, an anti-CD20agent, an anti-HLA-DR agent, an anti-HLA class I agent, an anti-CD52agent, an anti-A33 agent, an anti-GD3 agent, an anti-PSMA agent, ananti-Ceacan 1 agent, an anti-Galedin 9 agent, an anti-HVEM agent, ananti-VISTA agent, an anti-B7 H4 agent, an anti-HHLA2 agent, ananti-CD155 agent, an anti-CD80 agent, an anti-BTLA agent, an anti-CD160agent, an anti-CD28 agent, an anti-CD226 agent, an anti-CEACAM1 agent,an anti-TIM3 agent, an anti-TIGIT agent, an anti-CD96 agent, ananti-CD70 agent, an anti-CD27 agent, an anti-LIGHT agent, an anti-CD137agent, an anti-DR4 agent, an anti-CR5 agent, an anti-TNFRS agent, ananti-TNFR1 agent, an anti-FAS agent, an anti-CD95 agent, an anti-TRAILagent, an anti-DR6 agent, an anti-EDAR agent, an anti-NGFR agent, ananti-OPG agent, an anti-RANKL agent, an anti-LTβ receptor agent, ananti-BCMA agent, an anti-TACI agent, an anti-BAFFR agent, an anti-EDAR2agent, an anti-TROY agent, and an anti-RELT agent. For example, theimmunotherapy agent may be an anti-CTLA-4 agent, an anti-PD-1 agent, oran anti-PD-L1 agent.

In some embodiments, the immunotherapy agent is selected from the groupconsisting of an anti-CTLA-4 antibody or antigen-binding fragmentthereof, an anti-PD-1 antibody or antigen-binding fragment thereof, ananti-PD-L1 antibody or antigen-binding fragment thereof, an anti-PD-L2antibody or antigen-binding fragment thereof, a TNF-α cross-linkingantibody or antigen-binding fragment thereof, a TRAIL cross-linkingantibody or antigen-binding fragment thereof, an anti-CD27 antibody orantigen-binding fragment thereof, an anti-CD30 antibody orantigen-binding fragment thereof, an anti-CD40 antibody orantigen-binding fragment thereof, an anti-4-1 BB antibody orantigen-binding fragment thereof, an anti-GITR antibody orantigen-binding fragment thereof, an anti-OX40 antibody orantigen-binding fragment thereof, an anti-TRAILR1 antibody orantigen-binding fragment thereof, an anti-TRAILR2 antibody orantigen-binding fragment thereof, an anti-TWEAK antibody orantigen-binding fragment thereof, an anti-TWEAKR antibody orantigen-binding fragment thereof, an anti-cell surface lymphocyteprotein antibody or antigen-binding fragment thereof, an anti-BRAFantibody or antigen-binding fragment thereof, an anti-MEK antibody orantigen-binding fragment thereof, an anti-CD33 antibody orantigen-binding fragment thereof, an anti-CD20 antibody orantigen-binding fragment thereof, an anti-HLA-DR antibody orantigen-binding fragment thereof, an anti-HLA class I antibody orantigen-binding fragment thereof, an anti-CD52 antibody orantigen-binding fragment thereof, an anti-A33 antibody orantigen-binding fragment thereof, an anti-GD3 antibody orantigen-binding fragment thereof, an anti-PSMA antibody orantigen-binding fragment thereof, an anti-Ceacan 1 antibody orantigen-binding fragment thereof, an anti-Galedin 9 antibody orantigen-binding fragment thereof, an anti-HVEM antibody orantigen-binding fragment thereof, an anti-VISTA antibody orantigen-binding fragment thereof, an anti-B7 H4 antibody orantigen-binding fragment thereof, an anti-HHLA2 antibody orantigen-binding fragment thereof, an anti-CD155 antibody orantigen-binding fragment thereof, an anti-CD80 antibody orantigen-binding fragment thereof, an anti-BTLA antibody orantigen-binding fragment thereof, an anti-CD160 antibody orantigen-binding fragment thereof, an anti-CD28 antibody orantigen-binding fragment thereof, an anti-CD226 antibody orantigen-binding fragment thereof, an anti-CEACAM1 antibody orantigen-binding fragment thereof, an anti-TIM3 antibody orantigen-binding fragment thereof, an anti-TIGIT antibody orantigen-binding fragment thereof, an anti-CD96 antibody orantigen-binding fragment thereof, an anti-CD70 antibody orantigen-binding fragment thereof, an anti-CD27 antibody orantigen-binding fragment thereof, an anti-LIGHT antibody orantigen-binding fragment thereof, an anti-CD137 antibody orantigen-binding fragment thereof, an anti-DR4 antibody orantigen-binding fragment thereof, an anti-CR5 antibody orantigen-binding fragment thereof, an anti-TNFRS antibody orantigen-binding fragment thereof, an anti-TNFR1 antibody orantigen-binding fragment thereof, an anti-FAS antibody orantigen-binding fragment thereof, an anti-CD95 antibody orantigen-binding fragment thereof, an anti-TRAIL antibody orantigen-binding fragment thereof, an anti-DR6 antibody orantigen-binding fragment thereof, an anti-EDAR antibody orantigen-binding fragment thereof, an anti-NGFR antibody orantigen-binding fragment thereof, an anti-OPG antibody orantigen-binding fragment thereof, an anti-RANKL antibody orantigen-binding fragment thereof, an anti-LTβ receptor antibody orantigen-binding fragment thereof, an anti-BCMA antibody orantigen-binding fragment thereof, an anti-TACI antibody orantigen-binding fragment thereof, an anti-BAFFR antibody orantigen-binding fragment thereof, an anti-EDAR2 antibody orantigen-binding fragment thereof, an anti-TROY antibody orantigen-binding fragment thereof, and an anti-RELT antibody orantigen-binding fragment thereof. For example, the immunotherapy agentmay be an anti-CTLA-4 antibody or antigen-binding fragment thereof, ananti-PD-1 antibody or antigen-binding fragment thereof, or an anti-PD-L1antibody or antigen-binding fragment thereof.

In some embodiments, the pharmaceutical composition contains ananti-CTLA-4 antibody or antigen-binding fragment thereof, such asipilimumab or tremelimumab. Additionally or alternatively, thepharmaceutical composition may contain an anti-PD-1 antibody orantigen-binding fragment thereof, such as nivolumab, pembrolizumab,avelumab, durvalumab, or atezolizumab.

In some embodiments, the immunotherapy agent is an anti-cell surfacelymphocyte protein antibody or antigen-binding fragment thereof, such asan antibody or antigen-binding fragment thereof that binds one or moreof CD1, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11, CD12, CD13,CD14, CD15, CD16, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25,CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37,CD38, CD39, CD40, CD41, CD42, CD43, CD44, CD45, CD46, CD47, CD48, CD49,CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60, CD61,CD62, CD63, CD64, CD65, CD66, CD67, CD68, CD69, CD70, CD71, CD72, CD73,CD74, CD75, CD76, CD77, CD78, CD79, CD80, CD81, CD82, CD83, CD84, CD85,CD86, CD87, CD88, CD89, CD90, CD91, CD92, CD93, CD94, CD95, CD96, CD97,CD98, CD99, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107,CD108, CD109, CD110, CD111, CD112, CD113, CD114, CD115, CD116, CD117,CD118, CD119, CD120, CD121, CD122, CD123, CD124, CD125, CD126, CD127,CD128, CD129, CD130, CD131, CD132, CD133, CD134, CD135, CD136, CD137,CD138, CD139, CD140, CD141, CD142, CD143, CD144, CD145, CD146, CD147,CD148, CD149, CD150, CD151, CD152, CD153, CD154, CD155, CD156, CD157,CD158, CD159, CD160, CD161, CD162, CD163, CD164, CD165, CD166, CD167,CD168, CD169, CD170, CD171, CD172, CD173, CD174, CD175, CD176, CD177,CD178, CD179, CD180, CD181, CD182, CD183, CD184, CD185, CD186, CD187,CD188, CD189, CD190, CD191, CD192, CD193, CD194, CD195, CD196, CD197,CD198, CD199, CD200, CD201, CD202, CD203, CD204, CD205, CD206, CD207,CD208, CD209, CD210, CD211, CD212, CD213, CD214, CD215, CD216, CD217,CD218, CD219, CD220, CD221, CD222, CD223, CD224, CD225, CD226, CD227,CD228, CD229, CD230, CD231, CD232, CD233, CD234, CD235, CD236, CD237,CD238, CD239, CD240, CD241, CD242, CD243, CD244, CD245, CD246, CD247,CD248, CD249, CD250, CD251, CD252, CD253, CD254, CD255, CD256, CD257,CD258, CD259, CD260, CD261, CD262, CD263, CD264, CD265, CD266, CD267,CD268, CD269, CD270, CD271, CD272, CD273, CD274, CD275, CD276, CD277,CD278, CD279, CD280, CD281, CD282, CD283, CD284, CD285, CD286, CD287,CD288, CD289, CD290, CD291, CD292, CD293, CD294, CD295, CD296, CD297,CD298, CD299, CD300, CD301, CD302, CD303, CD304, CD305, CD306, CD307,CD308, CD309, CD310, CD311, CD312, CD313, CD314, CD315, CD316, CD317,CD318, CD319, and/or CD320.

In some embodiments, the immunotherapy agent is an agent (e.g., apolypeptide, antibody, antigen-binding fragment thereof, a single-chainpolypeptide, or construct thereof) that binds a chemokine or lymphokine,such as a chemokine or lymphokine involved in tumor growth. Forinstance, the immunotherapy agent may be an agent (e.g., polypeptide,antibody, antigen-binding fragment thereof, single-chain polypeptide, orconstruct thereof) that bind and inhibits the activity of one or more,or all, of CXCL1, CXCL2, CXCL3, CXCL8, CCL2 and CCL5. In someembodiments, the immunotherapy agent is an agent (e.g., a polypeptide,antibody, antigen-binding fragment thereof, a single-chain polypeptide,or construct thereof) that binds and inhibits the activity of one ormore, or all, of CCL3, CCL4, CCL8, and CCL22.

The immunotherapy agent may be capable of specifically binding one ormore of the immunological targets described in Table 1 of Mahoney etal., Cancer Immunotherapy, 14:561-584 (2015), the disclosure of which isincorporated herein by reference in its entirety. For example, theimmunotherapy agent may be an agent, such as an antibody orantigen-binding fragment thereof, that specifically binds one or more ofOX40L, TL1A, CD40L, LIGHT, BTLA, LAG3, TIM3, Singlecs, ICOS, B7-H3,B7-H4, VISTA, TMIGD2, BTNL2, CD48, KIR, LIR, LIR antibody, ILT, NKG2D,NKG2A, MICA, MICB, CD244, CSF1R, IDO, TGFβ, CD39, CD73, CXCR4, CXCL12,SIRPA, CD47, VEGF, or neuropilin.

In some embodiments, the immunotherapy agent is Targretin,Interferon-alpha, clobetasol, Peg Interferon (e.g., PEGASYS®),prednisone, Romidepsin, Bexarotene, methotrexate, Triamcinolone cream,anti-chemokines, Vorinostat, gabapentin, antibodies to lymphoid cellsurface receptors and/or lymphokines, antibodies to surface cancerproteins, and/or small molecular therapies like Vorinostat.

In some embodiments, the pharmaceutical composition contains abispecific antibody, such as a bispecific monoclonal antibody, in whichone arm of the antibody specifically binds TNFR2 and the otherspecifically binds an immune checkpoint protein, such as PD-1, PD-L1, orCTLA-4, among others described herein. The arm of the bispecificantibody that specifically binds TNFR2 may specifically bind, forexample, an epitope of human TNFR2 defined by one or more amino acidswithin CRD3 and/or an epitope defined by one or more amino acids withinCRD4, such as an epitope on human TNFR2 described above and herein asgiving rise to an antagonistic (e.g., a dominant antagonistic)phenotype.

In some embodiments, the bispecific antibody contains one arm thatspecifically binds TNFR2, such as an epitope of human TNFR2 describedabove, and one arm that specifically binds an immune checkpoint proteinspecifically binds PD-1. In some embodiments, the arm of the bispecificantibody that specifically binds PD-1 may specifically bind the sameepitope(s) on PD-1 as nivolumab, pembrolizumab, avelumab, durvalumab, oratezolizumab. For example, the arm of the bispecific antibody thatspecifically binds PD-1 may competitively inhibit the binding of PD-1 tonivolumab, pembrolizumab, avelumab, durvalumab, and/or atezolizumab, asassessed, for example, using a competitive binding assay describedherein or know in the art, such as a competitive ELISA.

In some embodiments, the bispecific antibody contains one arm thatspecifically binds TNFR2, such as an epitope of human TNFR2 describedabove, and one arm that specifically binds PD-L1. In some embodiments,the arm of the bispecific antibody that specifically binds PD-L1 mayspecifically bind the same epitope(s) on PD-L1 as atezolizumab oravelumab. For example, the arm of the bispecific antibody thatspecifically binds PD-L1 may competitively inhibit the binding of PD-L1to atezolizumab and/or avelumab, for example, using a competitivebinding assay described herein or know in the art, such as a competitiveELISA.

In some embodiments, the bispecific antibody contains one arm thatspecifically binds TNFR2, such as an epitope of human TNFR2 describedabove, and one arm that specifically binds CTLA-4. In some embodiments,the arm of the bispecific antibody that specifically binds CTLA-4 mayspecifically bind the same epitope(s) on CTLA-4 as ipilimumab ortremelimumab. For example, the arm of the bispecific antibody thatspecifically binds CTLA-4 may competitively inhibit the binding ofCTLA-4 to ipilimumab and/or tremelimumab, as assessed, for example,using a competitive binding assay described herein or know in the art,such as a competitive ELISA.

In some embodiments, the additional therapeutic agent in thepharmaceutical composition is a chimeric antigen receptor (CAR-T) agent,a chemotherapeutic agent, a small molecule anti-cancer agent, or acancer vaccine.

In some embodiments, the additional therapeutic agent in thepharmaceutical composition is a chimeric antigen receptor (CAR-T) agent,such as a T cell engineered to express a T cell receptor thatspecifically binds one or more antigens expressed on the surface of acancer cell. The antibody or antigen-binding fragment thereof,single-chain polypeptide, construct, polynucleotide, vector, or hostcell described herein (e.g., a TNFR2 antagonist antibody orantigen-binding fragment thereof) may be formulated forco-administration with a CAR-T agent for instance, by admixing theantibody or antigen-binding fragment thereof, single-chain polypeptide,construct, polynucleotide, vector, or host cell with the CAR-T agent. Insome embodiments, the antibody or antigen-binding fragment thereof,single-chain polypeptide, construct, polynucleotide, vector, or hostcell is formulated for administration separately from thechemotherapeutic agent, such as by way of serial administration.

In some embodiments, the additional therapeutic agent in thepharmaceutical composition is a chemotherapeutic agent, such as achemotherapeutic agent described herein. The antibody or antigen-bindingfragment thereof, single-chain polypeptide, construct, polynucleotide,vector, or host cell described herein (e.g., a TNFR2 antagonist antibodyor antigen-binding fragment thereof) may be formulated forco-administration with a chemotherapeutic agent, for instance, byadmixing the antibody or antigen-binding fragment thereof, single-chainpolypeptide, construct, polynucleotide, vector, or host cell with thechemotherapeutic agent. In some embodiments, the antibody orantigen-binding fragment thereof, single-chain polypeptide, construct,polynucleotide, vector, or host cell is formulated for administrationseparately from the chemotherapeutic agent. In some embodiments, thechemotherapeutic agent is conjugated directly to the antibody orantigen-binding fragment thereof, single-chain polypeptide, construct,polynucleotide, vector, or host cell, for instance, using bond-formingtechniques described herein or known in the art.

In some embodiments, the additional therapeutic agent is a smallmolecule anti-cancer agent, such as a small molecule described in Imaiet al., Nature Reviews Cancer 6:714-727 (2006), the disclosure of whichis incorporated herein by reference.

In some embodiments, the additional therapeutic agent is a cancervaccine, such as a vaccine described in Palucka et al., Journal ofImmunology 186:1325-1331 (2011), the disclosure of which is incorporatedherein by reference.

A seventh aspect features a method of producing the polypeptide (e.g., asingle-chain polypeptide, antibody, antigen-binding fragment thereof, orconstruct thereof) of the first aspect and/or the construct of thesecond aspect or any embodiments thereof. The method may includeexpressing a polynucleotide encoding the polypeptide or construct in ahost cell (e.g., a host cell described herein) and recovering thepolypeptide from host cell medium.

An eighth aspect features a method of reducing or inhibiting an immuneresponse mediated by a T-reg cell in a mammal (e.g., a human) byadministering to the mammal the polypeptide (e.g., a single-chainpolypeptide, antibody, antigen-binding fragment thereof, or constructthereof) of the first aspect or any embodiments thereof, the constructof the second aspect or any embodiments thereof, the polynucleotide ofthe third aspect or any embodiments thereof, the vector of the fourthaspect or any embodiments thereof, the host cell of the fifth aspect orany embodiments thereof, and/or the pharmaceutical composition of thesixth aspect or any embodiments thereof.

A ninth aspect features a method of treating a cell proliferationdisorder in a mammal (e.g., a human) by administering to the mammal thepolypeptide (e.g., a single-chain polypeptide, antibody, antigen-bindingfragment thereof, or construct thereof) of the first aspect or anyembodiments thereof, the construct of the second aspect or anyembodiments thereof, the polynucleotide of the third aspect or anyembodiments thereof, the vector of the fourth aspect or any embodimentsthereof, the host cell of the fifth aspect or any embodiments thereof,and/or the pharmaceutical composition of the sixth aspect or anyembodiments thereof.

The cell proliferation disorder may be, for example, a cancer, such as acancer selected from the group consisting of leukemia, lymphoma, livercancer, bone cancer, lung cancer, brain cancer, bladder cancer,gastrointestinal cancer, breast cancer, cardiac cancer, cervical cancer,uterine cancer, head and neck cancer, gallbladder cancer, laryngealcancer, lip and oral cavity cancer, ocular cancer, melanoma, pancreaticcancer, prostate cancer, colorectal cancer, testicular cancer, andthroat cancer. In some embodiments, the cancer is selected from thegroup consisting of Hodgkin's lymphoma, cutaneous non-Hodgkin'slymphoma, T cell lymphoma, ovarian cancer, colon cancer, multiplemyeloma, renal cell carcinoma, skin cancer, lung cancer, liver cancer,endometrial cancer, a cancer of the hematopoietic or lymphatic system, acancer of the central nervous system, breast cancer, pancreatic cancer,stomach cancer, esophageal cancer, and a cancer of the uppergastrointestinal tract. In some embodiments, the cancer is selected fromthe group consisting of T cell lymphoma, ovarian cancer, and coloncancer.

In some embodiments, the cancer is selected from the group consisting ofacute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML),chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML),adrenocortical carcinoma, AIDS-related lymphoma, primary CNS lymphoma,anal cancer, appendix cancer, astrocytoma, atypical teratoid/rhabdoidtumor, basal cell carcinoma, bile duct cancer, extrahepatic cancer,ewing sarcoma family, osteosarcoma and malignant fibrous histiocytoma,central nervous system embryonal tumors, central nervous system germcell tumors, craniopharyngioma, ependymoma, bronchial tumors, burkittlymphoma, carcinoid tumor, primary lymphoma, chordoma, chronicmyeloproliferative neoplasms, colon cancer, extrahepatic bile ductcancer, ductal carcinoma in situ (DCIS), endometrial cancer, ependymoma,esophageal cancer, esthesioneuroblastoma, extracranial germ cell tumor,extragonadal germ cell tumor, fallopian tube cancer, fibroushistiocytoma of bone, gastrointestinal carcinoid tumor, gastrointestinalstromal tumors (GIST), testicular germ cell tumor, gestationaltrophoblastic disease, glioma, childhood brain stem glioma, hairy cellleukemia, hepatocellular cancer, langerhans cell histiocytosis, hodgkinlymphoma, hypopharyngeal cancer, islet cell tumors, pancreaticneuroendocrine tumors, wilms tumor and other childhood kidney tumors,langerhans cell histiocytosis, small cell lung cancer, cutaneous T-celllymphoma, intraocular melanoma, merkel cell carcinoma, mesothelioma,metastatic squamous neck cancer, midline tract carcinoma, multipleendocrine neoplasia syndromes, multiple myeloma/plasma cell neoplasm,myelodysplastic syndromes, nasal cavity and paranasal sinus cancer,nasopharyngeal cancer, neuroblastoma, non-hodgkin lymphoma (NHL),non-small cell lung cancer (NSCLC), epithelial ovarian cancer, germ cellovarian cancer, low malignant potential ovarian cancer, pancreaticneuroendocrine tumors, papillomatosis, paraganglioma, paranasal sinusand nasal cavity cancer, parathyroid cancer, penile cancer, pharyngealcancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma,primary peritoneal cancer, rectal cancer, renal cancer, retinoblastoma,rhabdomyosarcoma, salivary gland cancer, kaposi sarcoma,rhabdomyosarcoma, sézary syndrome, small intestine cancer, soft tissuesarcoma, throat cancer, thymoma and thymic carcinoma, thyroid cancer,transitional cell cancer of the renal pelvis and ureter, urethralcancer, endometrial uterine cancer, uterine sarcoma, vaginal cancer,vulvar cancer, and Waldenström macroglobulinemia.

A tenth aspect features a method of treating an infectious disease in amammal (e.g., a human) by administering to the mammal the polypeptide(e.g., a single-chain polypeptide, antibody, antigen-binding fragmentthereof, or construct thereof) of the first aspect or any embodimentsthereof, the construct of the second aspect or any embodiments thereof,the polynucleotide of the third aspect or any embodiments thereof, thevector of the fourth aspect or any embodiments thereof, the host cell ofthe fifth aspect or any embodiments thereof, and/or the pharmaceuticalcomposition of the sixth aspect or any embodiments thereof. Theinfectious disease may be, for example, caused by a virus, bacterium,fungus, and/or parasite.

In some embodiments, the infectious disease is caused by a virusselected from the group consisting of hepatitis C virus, Yellow fevervirus, Kadam virus, Kyasanur Forest disease virus, Langat virus, Omskhemorrhagic fever virus, Powassan virus, Royal Farm virus, Karshi virus,tick-borne encephalitis virus, Neudoerfl virus, Sofjin virus, Loupingill virus, Negishi virus, Meaban virus, Saumarez Reef virus, Tyuleniyvirus, Aroa virus, dengue virus, Kedougou virus, Cacipacore virus,Koutango virus, Japanese encephalitis virus, Murray Valley encephalitisvirus, St. Louis encephalitis virus, Usutu virus, West Nile virus,Yaounde virus, Kokobera virus, Bagaza virus, Ilheus virus, Israel turkeymeningoencephalo-myelitis virus, Ntaya virus, Tembusu virus, Zika virus,Banzi virus, Bouboui virus, Edge Hill virus, Jugra virus, Saboya virus,Sepik virus, Uganda S virus, Wesselsbron virus, yellow fever virus,Entebbe bat virus, Yokose virus, Apoi virus, Cowbone Ridge virus,Jutiapa virus, Modoc virus, Sal Vieja virus, San Perlita virus, Bukalasabat virus, Carey Island virus, Dakar bat virus, Montana myotisleukoencephalitis virus, Phnom Penh bat virus, Rio Bravo virus, Tamanabat virus, cell fusing agent virus, Ippy virus, Lassa virus, lymphocyticchoriomeningitis virus (LCMV), Mobala virus, Mopeia virus, Amaparivirus, Flexal virus, Guanarito virus, Junin virus, Latino virus, Machupovirus, Oliveros virus, Paraná virus, Pichinde virus, Pirital virus,Sabiá virus, Tacaribe virus, Tamiami virus, Whitewater Arroyo virus,Chapare virus, Lujo virus, Hantaan virus, Sin Nombre virus, Dugbe virus,Bunyamwera virus, Rift Valley fever virus, La Crosse virus, Californiaencephalitis virus, Crimean-Congo hemorrhagic fever (CCHF) virus, Ebolavirus, Marburg virus, Venezuelan equine encephalitis virus (VEE),Eastern equine encephalitis virus (EEE), Western equine encephalitisvirus (WEE), Sindbis virus, rubella virus, Semliki Forest virus, RossRiver virus, Barmah Forest virus, O'nyong'nyong virus, and thechikungunya virus, smallpox virus, monkeypox virus, vaccinia virus,herpes simplex virus, human herpes virus, cytomegalovirus (CMV),Epstein-Barr virus (EBV), Varicella-Zoster virus, Kaposi's sarcomaassociated-herpesvirus (KSHV), influenza virus, severe acute respiratorysyndrome (SARS) virus, rabies virus, vesicular stomatitis virus (VSV),human respiratory syncytial virus (RSV), Newcastle disease virus,hendravirus, nipahvirus, measles virus, rinderpest virus, caninedistemper virus, Sendai virus, human parainfluenza virus (e.g., 1, 2, 3,and 4), rhinovirus, mumps virus, poliovirus, human enterovirus (A, B, C,and D), hepatitis A virus, coxsackievirus, hepatitis B virus, humanpapilloma virus, adeno-associated virus, astrovirus, JC virus, BK virus,SV40 virus, Norwalk virus, rotavirus, human immunodeficiency virus(HIV), human T-lymphotropic virus Types I and II.

In some embodiments, the infectious disease is caused by a bacteriumbelonging to a genus selected from the group consisting of Salmonella,Streptococcus, Bacillus, Listeria, Corynebacterium, Nocardia, Neisseria,Actinobacter, Moraxella, Enterobacteriacece, Pseudomonas, Escherichia,Klebsiella, Serratia, Enterobacter, Proteus, Salmonella, Shigella,Yersinia, Haemophilus, Bordatella, Legionella, Pasteurella, Francisella,Brucella, Bartonella, Clostridium, Vibrio, Campylobacter, andStaphylococcus.

In some embodiments, the infectious disease is caused by a fungusselected from the group consisting of Aspergillus, Candida, Malassezia,Trichosporon, Fusarium, Acremonium, Rhizopus, Mucor, Pneumocystis, andAbsidia.

In some embodiments, the infectious disease is caused by a parasiteselected from the group consisting of Entamoeba hystolytica, Giardialamblia, Cryptosporidium muris, Trypanosomatida gambiense,Trypanosomatida rhodesiense, Trypanosomatida crusi, Leishmania mexicana,Leishmania braziliensis, Leishmania tropica, Leishmania donovani,Toxoplasma gondii, Plasmodium vivax, Plasmodium ovale, Plasmodiummalariae, Plasmodium falciparum, Trichomonas vaginalis, and Histomonasmeleagridis. Exemplary helminthic parasites include Richuris trichiura,Ascaris lumbricoides, Enterobius vermicularis, Ancylostoma duodenale,Necator americanus, Strongyloides stercoralis, Wuchereria bancrofti, andDracunculus medinensis, Schistosoma mansoni, Schistosoma haematobium,Schistosoma japonicum, Fasciola hepatica, Fasciola gigantica,Heterophyes, Paragonimus westermani, Taenia solium, Taenia saginata,Hymenolepis nana, and Echinococcus granulosus.

In some embodiments of the eighth, ninth, and/or tenth aspect, themethod further includes administering to the human an immunotherapyagent. The immunotherapy agent may be, e.g., selected from the groupconsisting of an anti-CTLA-4 agent, an anti-PD-1 agent, an anti-PD-L1agent, an anti-PD-L2 agent, a TNF-α cross-linking agent, a TRAILcross-linking agent, an anti-CD27 agent, an anti-CD30 agent, ananti-CD40 agent, an anti-4-1 BB agent, an anti-GITR agent, an anti-OX40agent, an anti-TRAILR1 agent, an anti-TRAILR2 agent, an anti-TWEAKagent, an anti-TWEAKR agent, an anti-cell surface lymphocyte proteinagent, an anti-BRAF agent, an anti-MEK agent, an anti-CD33 agent, ananti-CD20 agent, an anti-HLA-DR agent, an anti-HLA class I agent, ananti-CD52 agent, an anti-A33 agent, an anti-GD3 agent, an anti-PSMAagent, an anti-Ceacan 1 agent, an anti-Galedin 9 agent, an anti-HVEMagent, an anti-VISTA agent, an anti-B7 H4 agent, an anti-HHLA2 agent, ananti-CD155 agent, an anti-CD80 agent, an anti-BTLA agent, an anti-CD160agent, an anti-CD28 agent, an anti-CD226 agent, an anti-CEACAM1 agent,an anti-TIM3 agent, an anti-TIGIT agent, an anti-CD96 agent, ananti-CD70 agent, an anti-CD27 agent, an anti-LIGHT agent, an anti-CD137agent, an anti-DR4 agent, an anti-CR5 agent, an anti-TNFRS agent, ananti-TNFR1 agent, an anti-FAS agent, an anti-CD95 agent, an anti-TRAILagent, an anti-DR6 agent, an anti-EDAR agent, an anti-NGFR agent, ananti-OPG agent, an anti-RANKL agent, an anti-LTβ receptor agent, ananti-BCMA agent, an anti-TACI agent, an anti-BAFFR agent, an anti-EDAR2agent, an anti-TROY agent, and an anti-RELT agent, such as ananti-CTLA-4 agent, an anti-PD-1 agent, and/or an anti-PD-L1 agent.

The immunotherapy agent administered to the human may be, for example,selected from the group consisting of an anti-CTLA-4 antibody orantigen-binding fragment thereof, an anti-PD-1 antibody orantigen-binding fragment thereof, an anti-PD-L1 antibody orantigen-binding fragment thereof, an anti-PD-L2 antibody orantigen-binding fragment thereof, a TNF-α cross-linking antibody orantigen-binding fragment thereof, a TRAIL cross-linking antibody orantigen-binding fragment thereof, an anti-CD27 antibody orantigen-binding fragment thereof, an anti-CD30 antibody orantigen-binding fragment thereof, an anti-CD40 antibody orantigen-binding fragment thereof, an anti-4-1 BB antibody orantigen-binding fragment thereof, an anti-GITR antibody orantigen-binding fragment thereof, an anti-OX40 antibody orantigen-binding fragment thereof, an anti-TRAILR1 antibody orantigen-binding fragment thereof, an anti-TRAILR2 antibody orantigen-binding fragment thereof, an anti-TWEAK antibody orantigen-binding fragment thereof, an anti-TWEAKR antibody orantigen-binding fragment thereof, an anti-cell surface lymphocyteprotein antibody or antigen-binding fragment thereof, an anti-BRAFantibody or antigen-binding fragment thereof, an anti-MEK antibody orantigen-binding fragment thereof, an anti-CD33 antibody orantigen-binding fragment thereof, an anti-CD20 antibody orantigen-binding fragment thereof, an anti-HLA-DR antibody orantigen-binding fragment thereof, an anti-HLA class I antibody orantigen-binding fragment thereof, an anti-CD52 antibody orantigen-binding fragment thereof, an anti-A33 antibody orantigen-binding fragment thereof, an anti-GD3 antibody orantigen-binding fragment thereof, an anti-PSMA antibody orantigen-binding fragment thereof, an anti-Ceacan 1 antibody orantigen-binding fragment thereof, an anti-Galedin 9 antibody orantigen-binding fragment thereof, an anti-HVEM antibody orantigen-binding fragment thereof, an anti-VISTA antibody orantigen-binding fragment thereof, an anti-B7 H4 antibody orantigen-binding fragment thereof, an anti-HHLA2 antibody orantigen-binding fragment thereof, an anti-CD155 antibody orantigen-binding fragment thereof, an anti-CD80 antibody orantigen-binding fragment thereof, an anti-BTLA antibody orantigen-binding fragment thereof, an anti-CD160 antibody orantigen-binding fragment thereof, an anti-CD28 antibody orantigen-binding fragment thereof, an anti-CD226 antibody orantigen-binding fragment thereof, an anti-CEACAM1 antibody orantigen-binding fragment thereof, an anti-TIM3 antibody orantigen-binding fragment thereof, an anti-TIGIT antibody orantigen-binding fragment thereof, an anti-CD96 antibody orantigen-binding fragment thereof, an anti-CD70 antibody orantigen-binding fragment thereof, an anti-CD27 antibody orantigen-binding fragment thereof, an anti-LIGHT antibody orantigen-binding fragment thereof, an anti-CD137 antibody orantigen-binding fragment thereof, an anti-DR4 antibody orantigen-binding fragment thereof, an anti-CR5 antibody orantigen-binding fragment thereof, an anti-TNFRS antibody orantigen-binding fragment thereof, an anti-TNFR1 antibody orantigen-binding fragment thereof, an anti-FAS antibody orantigen-binding fragment thereof, an anti-CD95 antibody orantigen-binding fragment thereof, an anti-TRAIL antibody orantigen-binding fragment thereof, an anti-DR6 antibody orantigen-binding fragment thereof, an anti-EDAR antibody orantigen-binding fragment thereof, an anti-NGFR antibody orantigen-binding fragment thereof, an anti-OPG antibody orantigen-binding fragment thereof, an anti-RANKL antibody orantigen-binding fragment thereof, an anti-LTβ receptor antibody orantigen-binding fragment thereof, an anti-BCMA antibody orantigen-binding fragment thereof, an anti-TACI antibody orantigen-binding fragment thereof, an anti-BAFFR antibody orantigen-binding fragment thereof, an anti-EDAR2 antibody orantigen-binding fragment thereof, an anti-TROY antibody orantigen-binding fragment thereof, and an anti-RELT antibody orantigen-binding fragment thereof. In some embodiments, the immunotherapyagent administered to the human is an anti-CTLA-4 antibody orantigen-binding fragment thereof, an anti-PD-1 antibody orantigen-binding fragment thereof, or an anti-PD-L1 antibody orantigen-binding fragment thereof.

In some embodiments of the eighth, ninth, and/or tenth aspect, themethod includes administering to the mammal (e.g., a human) ananti-CTLA-4 antibody or antigen-binding fragment thereof, such asipilimumab or tremelimumab. Additionally or alternatively, the methodmay include administering to the human an anti-PD-1 antibody orantigen-binding fragment thereof, such as nivolumab, pembrolizumab,avelumab, durvalumab, or atezolizumab.

In some embodiments of the eighth, ninth, and/or tenth aspect, themethod includes administering to the mammal (e.g., a human) an anti-cellsurface lymphocyte protein antibody or antigen-binding fragment thereof,such as an antibody or antigen-binding fragment thereof that binds oneor more of CD1, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11,CD12, CD13, CD14, CD15, CD16, CD17, CD18, CD19, CD20, CD21, CD22, CD23,CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35,CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD43, CD44, CD45, CD46, CD47,CD48, CD49, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59,CD60, CD61, CD62, CD63, CD64, CD65, CD66, CD67, CD68, CD69, CD70, CD71,CD72, CD73, CD74, CD75, CD76, CD77, CD78, CD79, CD80, CD81, CD82, CD83,CD84, CD85, CD86, CD87, CD88, CD89, CD90, CD91, CD92, CD93, CD94, CD95,CD96, CD97, CD98, CD99, CD100, CD101, CD102, CD103, CD104, CD105, CD106,CD107, CD108, CD109, CD110, CD111, CD112, CD113, CD114, CD115, CD116,CD117, CD118, CD119, CD120, CD121, CD122, CD123, CD124, CD125, CD126,CD127, CD128, CD129, CD130, CD131, CD132, CD133, CD134, CD135, CD136,CD137, CD138, CD139, CD140, CD141, CD142, CD143, CD144, CD145, CD146,CD147, CD148, CD149, CD150, CD151, CD152, CD153, CD154, CD155, CD156,CD157, CD158, CD159, CD160, CD161, CD162, CD163, CD164, CD165, CD166,CD167, CD168, CD169, CD170, CD171, CD172, CD173, CD174, CD175, CD176,CD177, CD178, CD179, CD180, CD181, CD182, CD183, CD184, CD185, CD186,CD187, CD188, CD189, CD190, CD191, CD192, CD193, CD194, CD195, CD196,CD197, CD198, CD199, CD200, CD201, CD202, CD203, CD204, CD205, CD206,CD207, CD208, CD209, CD210, CD211, CD212, CD213, CD214, CD215, CD216,CD217, CD218, CD219, CD220, CD221, CD222, CD223, CD224, CD225, CD226,CD227, CD228, CD229, CD230, CD231, CD232, CD233, CD234, CD235, CD236,CD237, CD238, CD239, CD240, CD241, CD242, CD243, CD244, CD245, CD246,CD247, CD248, CD249, CD250, CD251, CD252, CD253, CD254, CD255, CD256,CD257, CD258, CD259, CD260, CD261, CD262, CD263, CD264, CD265, CD266,CD267, CD268, CD269, CD270, CD271, CD272, CD273, CD274, CD275, CD276,CD277, CD278, CD279, CD280, CD281, CD282, CD283, CD284, CD285, CD286,CD287, CD288, CD289, CD290, CD291, CD292, CD293, CD294, CD295, CD296,CD297, CD298, CD299, CD300, CD301, CD302, CD303, CD304, CD305, CD306,CD307, CD308, CD309, CD310, CD311, CD312, CD313, CD314, CD315, CD316,CD317, CD318, CD319, and/or CD320.

In some embodiments of the eighth, ninth, and/or tenth aspect, themethod includes administering to the mammal (e.g., a human) an agent(e.g., a polypeptide, antibody, antigen-binding fragment thereof, asingle-chain polypeptide, or construct thereof) that binds a chemokineor lymphokine, such as a chemokine or lymphokine involved in tumorgrowth. For instance, the immunotherapy agent may be an agent (e.g.,polypeptide, antibody, antigen-binding fragment thereof, single-chainpolypeptide, or construct thereof) that bind and inhibits the activityof one or more, or all, of CXCL1, CXCL2, CXCL3, CXCL8, CCL2 and CCL5. Insome embodiments, the immunotherapy agent is an agent (e.g., apolypeptide, antibody, antigen-binding fragment thereof, a single-chainpolypeptide, or construct thereof) that binds and inhibits the activityof one or more, or all, of CCL3, CCL4, CCL8, and CCL22.

In some embodiments of the eighth, ninth, and/or tenth aspect, themethod includes administering to the mammal (e.g., a human) animmunotherapy agent capable of specifically binding one or more of theimmunological targets described in Table 1 of Mahoney et al., CancerImmunotherapy, 14:561-584 (2015), the disclosure of which isincorporated herein by reference in its entirety. For example, theimmunotherapy agent may be an agent, such as an antibody orantigen-binding fragment thereof, that specifically binds one or more ofOX40L, TL1A, CD40L, LIGHT, BTLA, LAG3, TIM3, Singlecs, ICOS, B7-H3,B7-H4, VISTA, TMIGD2, BTNL2, CD48, KIR, LIR, LIR antibody, ILT, NKG2D,NKG2A, MICA, MICB, CD244, CSF1R, IDO, TGFβ, CD39, CD73, CXCR4, CXCL12,SIRPA, CD47, VEGF, or neuropilin.

In some embodiments of the eighth, ninth, and/or tenth aspect, themethod includes administering to the mammal (e.g., a human) animmunotherapy agent selected from the group consisting of Targretin,Interferon-alpha, clobetasol, Peg Interferon (e.g., PEGASYS®),prednisone, Romidepsin, Bexarotene, methotrexate, Triamcinolone cream,anti-chemokines, Vorinostat, gabapentin, antibodies to lymphoid cellsurface receptors and/or lymphokines, antibodies to surface cancerproteins, and small molecular therapies like Vorinostat.

In some embodiments, the method includes administering to the mammal(e.g., a human) a CAR-T agent, a chemotherapeutic agent, a smallmolecule anti-cancer agent, or a cancer vaccine, such as a CAR-T agent,chemotherapeutic agent, small molecule anti-cancer agent, or cancervaccine described above and herein.

In some embodiments, the polypeptide, such as the single-chainpolypeptide, antibody, antigen-binding fragment thereof, or construct,which specifically binds TNFR2 is administered to the mammal (e.g., ahuman) in an amount of from about 0.001 mg/kg to about 100 mg/kg, suchas in an amount of from about 0.01 mg/kg to about 10 mg/kg.

An eleventh aspect features a kit containing the polypeptide (e.g., asingle-chain polypeptide, antibody, antigen-binding fragment thereof, orconstruct thereof) of the first aspect or any embodiments thereof, theconstruct of the second aspect or any embodiments thereof, thepolynucleotide of the third aspect or any embodiments thereof, thevector of the fourth aspect or any embodiments thereof, the host cell ofthe fifth aspect or any embodiments thereof, and/or the pharmaceuticalcomposition of the sixth aspect or any embodiments thereof.

In some embodiments, the kit contains instructions for transfecting thevector into a host cell. Additionally or alternatively, the kit maycontain instructions for expressing the polypeptide (e.g., asingle-chain polypeptide, antibody, antigen-binding fragment thereof, orconstruct thereof) in the host cell. The kit may include a reagent thatcan be used to express the polypeptide (e.g., a single-chainpolypeptide, antibody, antigen-binding fragment thereof, or constructthereof) in the host cell. In some embodiments, the kit includesinstructions for administering the agent to a mammal (e.g., a human),such as a human patient suffering from a cell proliferation disorderand/or an infectious disease described herein. In some embodiments, thekit contains instructions for making or using the agent.

Definitions

As used herein, the term “about” refers to a value that is no more than10% above or below the value being described. For example, the term“about 5 nM” indicates a range of from 4.5 nM to 5.5 nM.

As used herein, the term “antibody” (Ab) refers to an immunoglobulinmolecule that specifically binds to, or is immunologically reactivewith, a particular antigen, and includes polyclonal, monoclonal,genetically engineered and otherwise modified forms of antibodies,including but not limited to chimeric antibodies, humanized antibodies,primatized antibodies, heteroconjugate antibodies (e.g., bi- tri- andquad-specific antibodies, diabodies, triabodies, and tetrabodies), andantigen-binding fragments of antibodies, including e.g., Fab′, F(ab′)₂,Fab, Fv, rIgG, and scFv fragments. Moreover, unless otherwise indicated,the term “monoclonal antibody” (mAb) is meant to include both intactmolecules, as well as, antibody fragments (such as, for example, Fab andF(ab′)₂ fragments) that are capable of specifically binding to a targetprotein. Fab and F(ab′)₂ fragments lack the Fc fragment of an intactantibody, clear more rapidly from the circulation of the animal, and mayhave less non-specific tissue binding than an intact antibody (see Wahlet al., J. Nucl. Med. 24:316, 1983; incorporated herein by reference).

The term “antigen-binding fragment,” as used herein, refers to one ormore fragments of an antibody that retain the ability to specificallybind to a target antigen. The antigen-binding function of an antibodycan be performed by fragments of a full-length antibody. The antibodyfragments can be a Fab, F(ab′)₂, scFv, SMIP, diabody, a triabody, anaffibody, a nanobody, an aptamer, or a domain antibody. Examples ofbinding fragments encompassed of the term “antigen-binding fragment” ofan antibody include, but are not limited to: (i) a Fab fragment, amonovalent fragment consisting of the V_(L), V_(H), C_(L), and C_(H)1domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the V_(H) and C_(H)1 domains; (iv) a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody, (v) a dAb including V_(H) and V_(L) domains; (vi) a dAbfragment (Ward et al., Nature 341:544-546, 1989), which consists of aV_(H) domain; (vii) a dAb which consists of a V_(H) or a V_(L) domain;(viii) an isolated complementarity determining region (CDR); and (ix) acombination of two or more isolated CDRs which may optionally be joinedby a synthetic linker. Furthermore, although the two domains of the Fvfragment, V_(L) and V_(H), are coded for by separate genes, they can bejoined, using recombinant methods, by a linker that enables them to bemade as a single protein chain in which the V_(L) and V_(H) regions pairto form monovalent molecules (known as single-chain Fv (scFv); see,e.g., Bird et al., Science 242:423-426, 1988, and Huston et al., Proc.Natl. Acad. Sci. USA 85:5879-5883, 1988). These antibody fragments canbe obtained using conventional techniques known to those of skill in theart, and the fragments can be screened for utility in the same manner asintact antibodies. Antigen-binding fragments can be produced byrecombinant DNA techniques, enzymatic or chemical cleavage of intactimmunoglobulins, or, in some embodiments, by chemical peptide synthesisprocedures known in the art.

As used herein, the terms “anti-tumor necrosis factor receptor 2antibody,” “TNFR2 antibody,” “anti-TNFR2 antibody portion,” and/or“anti-TNFR2 antibody fragment” and the like include any protein orpeptide-containing molecule that includes at least a portion of animmunoglobulin molecule, such as, but not limited, to at least onecomplementarity determining region (CDR) of a heavy or light chain or aligand-binding portion thereof, a heavy chain or light chain variableregion, a heavy chain or light chain constant region, or any portionthereof, that is capable of specifically binding to TNFR2. For instance,two or more portions of an immunoglobulin molecule may be covalentlybound to one another, e.g., via an amide bond, a thioether bond, acarbon-carbon bond, a disulfide bridge, or by a linker, such as a linkerdescribed herein or known in the art. TNFR2 antibodies also includeantibody-like protein scaffolds, such as the tenth fibronectin type IIIdomain (¹⁰Fn3), which contains BC, DE, and FG structural loops similarin structure and solvent accessibility to antibody CDRs. The tertiarystructure of the ¹⁰Fn3 domain resembles that of the variable region ofthe IgG heavy chain, and one of skill in the art can graft, e.g., theCDRs of a TNFR2 monoclonal antibody onto the fibronectin scaffold byreplacing residues of the BC, DE, and FG loops of ¹⁰Fn3 with residuesfrom the CDR-H1, CDR-H2, or CDR-H3 regions of a TNFR2 monoclonalantibody.

As used herein, the terms “antagonist TNFR2 antibody” and “antagonisticTNFR2 antibody” refer to TNFR2 antibodies that are capable of inhibitingor reducing activation of TNFR2, attenuating one or more signaltransduction pathways mediated by TNFR2, and/or reducing or inhibitingat least one activity mediated by activation of TNFR2. For example,antagonistic TNFR2 antibodies may inhibit or reduce the growth andproliferation of regulatory T cells. Antagonistic TNFR2 antibodies mayinhibit or reduce TNFR2 activation by blocking TNFR2 from binding TNFα.In this way, antagonistic TNFR2 antibodies may block the trimerizationof TNFR2 that would otherwise be induced by interacting with TNFα, thusresulting in suppression of TNFR2 activity.

As used herein, the term “bispecific antibodies” refers to antibodies(e.g., monoclonal, often human or humanized antibodies) that havebinding specificities for at least two different antigens. For example,one of the binding specificities can be directed towards TNFR2, theother can be for any other antigen, e.g., for a cell-surface protein,receptor, receptor subunit, tissue-specific antigen, virally derivedprotein, virally encoded envelope protein, bacterially derived protein,or bacterial surface protein, etc.

As used herein, the phrase “chemotherapeutic agent” refers to anychemical agent with therapeutic usefulness in the treatment of cancer,such as a cancer described herein. Chemotherapeutic agents encompassboth chemical and biological agents. These agents can function toinhibit a cellular activity upon which a cancer cell depends forcontinued survival. Categories of chemotherapeutic agents includealkylating/alkaloid agents, antimetabolites, hormones, hormone analogs,and antineoplastic drugs. Exemplary chemotherapeutic agents suitable foruse in conjunction with the compositions and methods described hereininclude, without limitation, those set forth in Slapak and Kufe,Principles of Cancer Therapy, Chapter 86 in Harrison's Principles ofInternal medicine, 14^(th) edition; Perry et al., Chemotherapeutic,Chapter 17 in Abeloff, Clinical Oncology 2^(nd) ed., 2000; Baltzer L.and Berkery R. (eds): Oncology Pocket Guide to Chemotherapeutic, 2^(nd)ed. St. Luois, mosby-Year Book, 1995; Fischer D. S., Knobf M. F.,Durivage H. J. (eds): The Cancer Chemotherapeutic Handbook, 4^(th) ed.St. Luois, Mosby-Year Handbook, the disclosures of each of which areincorporated herein by reference as they pertain to chemotherapeuticagents.

As used herein, the term “chimeric” antibody refers to an antibodyhaving variable domain sequences (e.g., CDR sequences) derived from animmunoglobulin of one source organism, such as rat or mouse, andconstant regions derived from an immunoglobulin of a different organism(e.g., a human, another primate, pig, goat, rabbit, hamster, cat, dog,guinea pig, member of the bovidae family (such as cattle, bison,buffalo, elk, and yaks, among others), cow, sheep, horse, or bison,among others). Methods for producing chimeric antibodies are known inthe art. See, e.g., Morrison, 1985, Science 229(4719): 1202-7; Oi et al,1986, BioTechniques 4:214-221; Gillies et al, 1985, J. Immunol. Methods125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397;incorporated herein by reference.

As used herein, the term “complementarity determining region” (CDR)refers to a hypervariable region found both in the light chain and theheavy chain variable domains. The more highly conserved portions ofvariable domains are called the framework regions (FRs). As isappreciated in the art, the amino acid positions that delineate ahypervariable region of an antibody can vary, depending on the contextand the various definitions known in the art. Some positions within avariable domain may be viewed as hybrid hypervariable positions in thatthese positions can be deemed to be within a hypervariable region underone set of criteria while being deemed to be outside a hypervariableregion under a different set of criteria. One or more of these positionscan also be found in extended hypervariable regions. The antibodiesdescribed herein may comprising modifications in these hybridhypervariable positions. The variable domains of native heavy and lightchains each comprise four framework regions that primarily adopt aβ-sheet configuration, connected by three CDRs, which form loops thatconnect, and in some cases form part of, the β-sheet structure. The CDRsin each chain are held together in close proximity by the FR regions inthe order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and, with the CDRs from theother antibody chains, contribute to the formation of the target bindingsite of antibodies (see Kabat et al, Sequences of Proteins ofImmunological Interest (National Institute of Health, Bethesda, Md.1987; incorporated herein by reference). As used herein, numbering ofimmunoglobulin amino acid residues is done according to theimmunoglobulin amino acid residue numbering system of Kabat et al,unless otherwise indicated.

As used herein, the terms “conservative mutation,” “conservativesubstitution,” or “conservative amino acid substitution” refer to asubstitution of one or more amino acids for one or more different aminoacids that exhibit similar physicochemical properties, such as polarity,electrostatic charge, and steric volume. These properties are summarizedfor each of the twenty naturally-occurring amino acids in table 2 below.

TABLE 2 Representative physicochemical properties of naturally-occurringamino acids Electrostatic 3 1 character at Letter Letter Side-chainphysiological Steric Amino Acid Code Code Polarity pH (7.4) Volume^(†)Alanine Ala A nonpolar neutral small Arginine Arg R polar cationic largeAsparagine Asn N polar neutral intermediate Aspartic acid Asp D polaranionic intermediate Cysteine Cys C nonpolar neutral intermediateGlutamic acid Glu E polar anionic intermediate Glutamine Gln Q polarneutral intermediate Glycine Gly G nonpolar neutral small Histidine HisH polar Both neutral large and cationic forms in equilibrium at pH 7.4Isoleucine Ile I nonpolar neutral large Leucine Leu L nonpolar neutrallarge Lysine Lys K polar cationic large Methionine Met M nonpolarneutral large Phenylalanine Phe F nonpolar neutral large Proline Pro Pnon-polar neutral intermediate Serine Ser S polar neutral smallThreonine Thr T polar neutral intermediate Tryptophan Trp W nonpolarneutral bulky Tyrosine Tyr Y polar neutral large Valine Val V nonpolarneutral intermediate ^(†)based on volume in A³: 50-100 is small, 100-150is intermediate, 150-200 is large, and >200 is bulky

From this table it is appreciated that the conservative amino acidfamilies include, e.g., (i) G, A, V, L, I, P, and M; (ii) D and E; (iii)C, S and T; (iv) H, K and R; (v) N and Q; and (vi) F, Y and W. Aconservative mutation or substitution is therefore one that substitutesone amino acid for a member of the same amino acid family (e.g., asubstitution of Ser for Thr or Lys for Arg).

Amino acid substitutions may be represented herein using the convention:(AA1)(N)(AA2), where “AA1” represents the amino acid normally present atparticular site within an amino acid sequence, “N” represents theresidue number within the amino acid sequence at which the substitutionoccurs, and “AA2” represents the amino acid present in the amino acidsequence after the substitution is effectuated. For example, thenotation “C232S” in the context of an antibody hinge region, such as anIgG2 antibody hinge region, refers to a substitution of thenaturally-occurring cysteine residue for a serine residue at amino acidresidue 232 of the indicated hinge amino acid sequence. Likewise, thenotation “C233S” in the context of an antibody hinge region, such as anIgG2 antibody hinge region, refers to a substitution of thenaturally-occurring cysteine residue for a serine residue at amino acidresidue 233 of the indicated hinge amino acid sequence.

As used herein, the term “conjugate” refers to a compound formed by thechemical bonding of a reactive functional group of one molecule with anappropriately reactive functional group of another molecule.

As used herein in the context of a TNFR2 antagonist, the term“construct” refers to a fusion protein containing a first polypeptidedomain bound to a second polypeptide domain. The polypeptide domains mayeach independently be antagonistic TNFR2 single chain polypeptides, forinstance, as described herein. The first polypeptide domain may becovalently bound to the second polypeptide domain, for instance, by wayof a linker, such as a peptide linker or a disulfide bridge, amongothers. Exemplary linkers that may be used to join the polypeptidedomains of an antagonistic TNFR2 construct include, without limitation,those that are described in Leriche et al., Bioorg. Med. Chem.,20:571-582 (2012), the disclosure of which is incorporated herein byreference in its entirety.

As used herein, the term “derivatized antibodies” refers to antibodiesthat are modified by a chemical reaction so as to cleave residues or addchemical moieties not native to an isolated antibody. Derivatizedantibodies can be obtained by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by addition of known chemicalprotecting/blocking groups, proteolytic cleavage, linkage to a cellularligand or other protein. Any of a variety of chemical modifications canbe carried out by known techniques, including, without limitation,specific chemical cleavage, acetylation, formylation, metabolicsynthesis of tunicamycin, etc. using established procedures.Additionally, the derivative can contain one or more non-natural aminoacids, e.g., using amber suppression technology (see, e.g., U.S. Pat.No. 6,964,859; incorporated herein by reference).

As used herein, the term “diabodies” refers to bivalent antibodiescomprising two polypeptide chains, in which each polypeptide chainincludes V_(H) and V_(L) domains joined by a linker that is too short(e.g., a linker composed of five amino acids) to allow forintramolecular association of VH and VL domains on the same peptidechain. This configuration forces each domain to pair with acomplementary domain on another polypeptide chain so as to form ahomodimeric structure. Accordingly, the term “triabodies” refers totrivalent antibodies comprising three peptide chains, each of whichcontains one VH domain and one VL domain joined by a linker that isexceedingly short (e.g., a linker composed of 1-2 amino acids) to permitintramolecular association of VH and VL domains within the same peptidechain. In order to fold into their native structure, peptides configuredin this way typically trimerize so as to position the VH and VL domainsof neighboring peptide chains spatially proximal to one another topermit proper folding (see Holliger et al., Proc. Natl. Acad. Sci. USA90:6444-48, 1993; incorporated herein by reference).

As used herein, a “disulfide-bonded isoform” of an antibody orantigen-binding fragment thereof is a form of the antibody orantigen-binding fragment thereof having a particular internal disulfidebonding pattern. Disulfide-bonded isoforms are structural isomers of agiven antibody or antigen-binding fragment thereof that do not differfrom one another in amino acid sequence but exhibit different disulfidebond connectivities. For example, in the context of a human IgG2antibody or variant thereof, the antibody may exist in one of fourpossible disulfide-bonded isoforms, represented herein as isoformsIgG2-A, IgG2-B, IgG2-A/B₁, and IgG2-A/B₂. The disulfide bondingconnectivities within each of these isoforms are shown graphically inFIGS. 13A-13D.

As used herein, a “dominant antagonist” of TNFR2 is an antagonist (e.g.,an antagonistic polypeptide, such as a single-chain polypeptide,antibody, or antigen-binding fragment thereof) that is capable ofinhibiting TNFR2 activation even in the presence of a TNFR2 agonist,such as TNFα, or IL-2. For example, a TNFR2 antagonist is a dominantantagonist if the IC₅₀ of the antagonist increases by less than 200%(e.g., less than 200%, 100%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%,10%, 5%, 1%, or less) in the presence of a TNFR2 agonist (e.g., TNFα) orIL-2 relative to the IC₅₀ of the antagonist as measured in the sameassay in the absence of a TNFR2 agonist, such as TNFα, or IL-2.Inhibition of TNFR2 activation can be assessed, for instance, bymeasuring the inhibition of proliferation of TNFR2+ cells, such as T-regcells, cancer cells that express TNFR2, or myeloid-derived suppressorcells, as well as by measuring the inhibition of NFκB signaling (e.g.,by monitoring the reduction in expression of one or more genes selectedfrom the group consisting of CHUK, NFKBIE, NFKBIA, MAP3K11, TRAF2,TRAF3, relB, and cIAP2/BIRC3 in a conventional gene expression assay).

As used herein, a “dual variable domain immunoglobulin” (“DVD-Ig”)refers to an antibody that combines the target-binding variable domainsof two monoclonal antibodies via linkers to create a tetravalent,dual-targeting single agent. (Gu et al., Meth. Enzymol., 502:25-41,2012; incorporated by reference herein). Suitable linkers for use in thelight chains of the DVDs described herein include those identified onTable 2.1 on page 30 of Gu et al.: the short K chain linkers ADAAP (SEQID NO: 118) (murine) and TVAAP (SEQ ID NO: 119) (human); the long κchain linkers ADAAPTVSIFP (SEQ ID NO: 120) (murine) and TVAAPSVFIFPP(SEQ ID NO: 121) (human); the short λ chain linker QPKAAP (SEQ ID NO:122) (human); the long λ chain linker QPKAAPSVTLFPP (SEQ ID NO: 123)(human); the GS-short linker GGSGG (SEQ ID NO: 124), the GS-mediumlinker GGSGGGGSG (SEQ ID NO: 125), and the GS-long linker GGSGGGGSGGGGS(SEQ ID NO: 126) (all GS linkers are murine and human). Suitable linkersfor use in the heavy chains of the DVDs include those identified onTable 2.1 on page 30 of Gu & Ghayur, 2012, Methods in Enzymology502:25-41, incorporated by reference herein: the short linkers AKTTAP(SEQ ID NO: 127) (murine) and ASTKGP (SEQ ID NO: 128) (human); the longlinkers AKTTAPSVYPLAP (SEQ ID NO: 129) (murine) and ASTKGPSVFPLAP (SEQID NO: 130) (human); the GS-short linker GGGGSG (SEQ ID NO: 131), theGS-medium linker GGGGSGGGGS (SEQ ID NO: 26), and the GS-long linkerGGGGSGGGGSGGGG (SEQ ID NO: 133) (all GS linkers are murine and human).

As used herein, the term “endogenous” describes a molecule (e.g., apolypeptide, nucleic acid, or cofactor) that is found naturally in aparticular organism (e.g., a human) or in a particular location withinan organism (e.g., an organ, a tissue, or a cell, such as a human cell).

As used herein, the term “epitope” refers to a portion of an antigenthat is recognized and bound by a polypeptide, such as an antibody,antigen-binding fragment thereof, single-chain polypeptide, or constructas described herein. In the context of a protein antigen (such as TNFR2,e.g., human TNFR2 designated by SEQ ID NO: 7 or TNFR2 of a non-humanmammal, such as a non-human mammal described herein), an epitope may bea continuous epitope, which is a single, uninterrupted segment of one ormore amino acids covalently linked to one another by peptide bonds inwhich all of the component amino acids bind the polypeptide (e.g.,antibody, antigen-binding fragment thereof, single-chain polypeptide, orconstruct thereof). Exemplary assays for determining the binding of anantagonistic TNFR2 polypeptide to specific amino acids within an antigenare described in Example 1, below. Continuous epitopes may be composed,for instance, of 1, 5, 10, 15, 20, or more amino acids within anantigen, such as a TNFR2 protein described herein (for instance, humanTNFR2 designated by SEQ ID NO: 7). For example, a continuous epitope maybe composed of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, or more amino acids within an antigen). Examplesof continuous epitopes on TNFR2 that are bound by antagonisticpolypeptides (e.g., single-chain polypeptides, antibodies,antigen-binding fragments thereof, and constructs thereof) describedherein include one or more continuous residues of, or all residues of,the SSTDICRPHQI motif (SEQ ID NO: 288), one or more continuous residuesof, or all residues of, the CALSKQEGCRLCAPL motif (SEQ ID NO: 289), andone or more continuous residues of, or all residues of, the TSDVVCKPCAmotif (SEQ ID NO: 290), as well as corresponding regions on TNFR2proteins of non-human mammals (e.g., bison, cattle, and others describedherein). In some embodiments, an epitope may be a discontinuous epitope,which contains two or more segments of amino acids each separated fromone another in an antigen's amino acid sequence by one or moreintervening amino acid residues. Discontinuous epitopes may be composed,for instance, of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more such segments ofamino acid residues, such as one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more) segments containing amino acids from within one or moreof the SSTDICRPHQI motif (SEQ ID NO: 288), the CALSKQEGCRLCAPL motif(SEQ ID NO: 289), and the TSDVVCKPCA motif (SEQ ID NO: 290) within humanTNFR2, as well as corresponding regions on TNFR2 proteins of non-humanmammals (e.g., bison, cattle, and others described herein). Despite thisseparation by intervening amino acids, the segments that compose adiscontinuous epitope may be, for instance, spatially proximal to oneanother in the three-dimensional conformation of the antigen. Exemplarydiscontinuous epitopes on TNFR2 that are bound by antagonisticpolypeptides (e.g., single-chain polypeptides, antibodies,antigen-binding fragments thereof, and constructs thereof) describedherein include epitopes containing the following elements: (i) one ormore residues, or all residues, of the SSTDICRPHQI motif (SEQ ID NO:288); (ii) one or more residues, or all residues, of the CALSKQEGCRLCAPLmotif (SEQ ID NO: 289), and (iii) one or more residues, r all residues,of the TSDVVCKPCA motif (SEQ ID 290). Additional examples ofdiscontinuous epitopes on TNFR2 that are bound by antagonisticpolypeptides (e.g., single-chain polypeptides, antibodies,antigen-binding fragments thereof, and constructs thereof) describedherein include epitopes containing elements (i) and (ii) above, epitopescontaining elements (i) and (iii) above, and epitopes containingelements (ii) and (iii) above.

As used herein, the term “exogenous” describes a molecule (e.g., apolypeptide, nucleic acid, or cofactor) that is not found naturally in aparticular organism (e.g., a human) or in a particular location withinan organism (e.g., an organ, a tissue, or a cell, such as a human cell).Exogenous materials include those that are provided from an externalsource to an organism or to cultured matter extracted there from.

As used herein, the term “framework region” or “FW region” includesamino acid residues that are adjacent to the CDRs. FW region residuesmay be present in, for example, human antibodies, rodent-derivedantibodies (e.g., murine antibodies), humanized antibodies, primatizedantibodies, chimeric antibodies, antibody fragments (e.g., Fabfragments), single-chain antibody fragments (e.g., scFv fragments),antibody domains, and bispecific antibodies, among others.

As used herein, the term “fusion protein” refers to a protein that isjoined via a covalent bond to another molecule. A fusion protein can bechemically synthesized by, e.g., an amide-bond forming reaction betweenthe N-terminus of one protein to the C-terminus of another protein.Alternatively, a fusion protein containing one protein covalently boundto another protein can be expressed recombinantly in a cell (e.g., aeukaryotic cell or prokaryotic cell) by expression of a polynucleotideencoding the fusion protein, for example, from a vector or the genome ofthe cell. A fusion protein may contain one protein that is covalentlybound to a linker, which in turn is covalently bound to anothermolecule. Examples of linkers that can be used for the formation of afusion protein include peptide-containing linkers, such as those thatcontain naturally occurring or non-naturally occurring amino acids. Insome embodiments, it may be desirable to include D-amino acids in thelinker, as these residues are not present in naturally-occurringproteins and are thus more resistant to degradation by endogenousproteases. Linkers can be prepared using a variety of strategies thatare well known in the art, and depending on the reactive components ofthe linker, can be cleaved by enzymatic hydrolysis, photolysis,hydrolysis under acidic conditions, hydrolysis under basic conditions,oxidation, disulfide reduction, nucleophilic cleavage, or organometalliccleavage (Leriche et al., Bioorg. Med. Chem., 20:571-582, 2012).

As used herein, the term “heterospecific antibodies” refers tomonoclonal, preferably human or humanized, antibodies that have bindingspecificities for at least two different antigens. Traditionally, therecombinant production of heterospecific antibodies is based on theco-expression of two immunoglobulin heavy chain-light chain pairs, wherethe two heavy chains have different specificities (Milstein et al.,Nature 305:537, 1983). Similar procedures are disclosed, e.g., in WO93/08829, U.S. Pat. Nos. 6,210,668; 6,193,967; 6,132,992; 6,106,833;6,060,285; 6,037,453; 6,010,902; 5,989,530; 5,959,084; 5,959,083;5,932,448; 5,833,985; 5,821,333; 5,807,706; 5,643,759, 5,601,819;5,582,996, 5,496,549, 4,676,980, WO 91/00360, WO 92/00373, EP 03089,Traunecker et al., EMBO J. 10:3655 (1991), Suresh et al., Methods inEnzymology 121:210 (1986); incorporated herein by reference.Heterospecific antibodies can include Fc mutations that enforce correctchain association in multi-specific antibodies, as described by Klein etal, mAbs 4(6):653-663, 2012; incorporated herein by reference.

As used herein, the term “hinge region” refers to the domain of anantibody or antigen-binding fragment thereof (e.g., an IgG2 antibody orantigen-binding fragment thereof) located between the antigen-bindingportion(s) of the antibody or antigen-binding fragment thereof, such asthe Fab region of the antibody or antigen-binding fragment thereof, andthe portion of the antibody or antigen-binding fragment thereof thatdictates the isotype of the antibody or antigen-binding fragmentthereof, such as the Fc region of the antibody or antigen-bindingfragment thereof. For example, in the context of a monoclonal antibody,the hinge region is the polypeptide situated approximately in the centerof each heavy chain, connecting the CH1 domain to the CH2 and CH3domains. The hinge region of an antibody or antigen-binding fragmentthereof may provide a chemical linkage between chains of the antibody orantigen-binding fragment thereof. For instance, in a monoclonalantibody, the cysteine residues within the hinge region form inter-chaindisulfide bonds, thereby providing explicit covalent bonds between heavychains. The amino acid sequence of wild-type human IgG2 is ERKCCVECPPCP(SEQ ID NO: 292). As used herein, antibody hinge regions are numberedaccording to the numbering system of Kabat et al, Sequences of Proteinsof Immunological Interest (National Institute of Health, Bethesda, Md.1987), the disclosure of which is incorporated herein by reference. Forexample, using the numbering scheme of Kabat et al, the wild-type humanIgG2 hinge region set forth in SEQ ID NO: 292 is numbered from residues226 to 243, such that the N-terminal glutamate residue of SEQ ID NO: 292is residue 226 and the C-terminal proline residue of SEQ ID NO: 292 isresidue 243. Throughout the present disclosure, variant IgG2 hingeregions, such as the variant set forth in SEQ ID NO: 291 (ERKCCVECPPCP),are numbered according to the convention of Kabat et al unlessexplicitly stated to the contrary.

As used herein, the term “human antibody” refers to an antibody in whichsubstantially every part of the protein (e.g., CDR, framework, C_(L),C_(H) domains (e.g., C_(H)1, C_(H)2, C_(H)3), hinge, (V_(L), V_(H))) issubstantially non-immunogenic in humans, with only minor sequencechanges or variations. A human antibody can be produced in a human cell(e.g., by recombinant expression), or by a non-human animal or aprokaryotic or eukaryotic cell that is capable of expressingfunctionally rearranged human immunoglobulin (e.g., heavy chain and/orlight chain) genes. Further, when a human antibody is a single-chainantibody, it can include a linker peptide that is not found in nativehuman antibodies. For example, an Fv can comprise a linker peptide, suchas two to about eight glycine or other amino acid residues, whichconnects the variable region of the heavy chain and the variable regionof the light chain. Such linker peptides are considered to be of humanorigin. Human antibodies can be made by a variety of methods known inthe art including phage display methods using antibody libraries derivedfrom human immunoglobulin sequences. See U.S. Pat. Nos. 4,444,887 and4,716,111; and PCT publications WO 1998/46645; WO 1998/50433; WO1998/24893; WO 1998/16654; WO 1996/34096; WO 1996/33735; and WO1991/10741; incorporated herein by reference. Human antibodies can alsobe produced using transgenic mice that are incapable of expressingfunctional endogenous immunoglobulins, but which can express humanimmunoglobulin genes. See, e.g., PCT publications WO 98/24893; WO92/01047; WO 96/34096; WO 96/33735; U.S. Pat. Nos. 5,413,923; 5,625,126;5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793;5,916,771; and 5,939,598; incorporated by reference herein.

As used herein, the term “humanized” antibodies refers to forms ofnon-human (e.g., murine) antibodies that are chimeric immunoglobulins,immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′,F(ab′)₂ or other target-binding subdomains of antibodies) which containminimal sequences derived from non-human immunoglobulin. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin. Allor substantially all of the FR regions may also be those of a humanimmunoglobulin sequence. The humanized antibody can also comprise atleast a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin consensus sequence. Methods of antibodyhumanization are known in the art. See, e.g., Riechmann et al., Nature332:323-7, 1988; U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,761;5,693,762; and 6,180,370 to Queen et al; EP239400; PCT publication WO91/09967; U.S. Pat. No. 5,225,539; EP592106; and EP519596; incorporatedherein by reference.

As used herein, the term “hydrophobic side-chain” refers to an aminoacid side-chain that exhibits low solubility in water relative due to,e.g., the steric or electronic properties of the chemical moietiespresent within the side-chain. Examples of amino acids containinghydrophobic side-chains include those containing unsaturated aliphatichydrocarbons, such as alanine, valine, leucine, isoleucine, proline, andmethionine, as well as amino acids containing aromatic ring systems thatare electrostatically neutral at physiological pH, such as tryptophan,phenylalanine, and tyrosine.

As used herein, the term “immunotherapy agent” refers to a compound,such as an antibody, antigen-binding fragment thereof, single-chainpolypeptide, or construct as described herein, that specifically bindsan immune checkpoint protein (e.g., immune checkpoint receptor orligand) and exerts an antagonistic effect on the receptor or ligand,thereby reducing or inhibiting the signal transduction of the receptoror ligand that would otherwise lead to a downregulation of the immuneresponse. Immunotherapy agents include compounds, such as antibodies,antigen-binding fragments, single-chain polypeptides, and constructs,capable of specifically binding receptors expressed on the surfaces ofhematopoietic cells, such as lymphocytes (e.g., T cells), andsuppressing the signaling induced by the receptor or ligand that wouldotherwise lead to tolerance towards an endogenous (“self”) antigen, suchas a tumor-associated antigen. Immunotherapy agents may reduce thesignaling induced by the receptor or ligand by, for example, 1%, 2%, 3%,4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% relative to the signaling induced by the receptor orligand exhibited in the absence of the immunotherapy agent. Exemplaryassays that can be used to measure the extent of receptor or ligandsignaling include, for example, enzyme-linked immunosorbant assay(ELISA) techniques to measure protein expression alterations that areassociated with a particular signal transduction pathway, as well aspolymerase chain reaction (PCR)-based techniques, such as quantitativePCR, reverse-transcription PCR, and real-time PCR experiments useful fordetermining changes in gene expression associated with a particularsignal transduction pathway, among others. Exemplary methods that can beused to determine whether an agent is an “immunotherapy agent” includethe assays described in Mahoney et al., Cancer Immunotherapy, 14:561-584(2015), the disclosure of which is incorporated herein by reference inits entirety. Examples of immunotherapy agents include, e.g., antibodiesor antigen-binding fragments thereof that specifically bind one or moreof OX40L, TL1A, CD40L, LIGHT, BTLA, LAG3, TIM3, Singlecs, ICOS, B7-H3,B7-H4, VISTA, TMIGD2, BTNL2, CD48, KIR, LIR, LIR antibody, ILT, NKG2D,NKG2A, MICA, MICB, CD244, CSF1R, IDO, TGFβ, CD39, CD73, CXCR4, CXCL12,SIRPA, CD47, VEGF, and neuropilin. Additional example of immunotherapyagents include Targretin, Interferon-alpha, clobetasol, Peg Interferon(e.g., PEGASYS®), prednisone, Romidepsin, Bexarotene, methotrexate,Triamcinolone cream, anti-chemokines, Vorinostat, gabapentin, antibodiesto lymphoid cell surface receptors and/or lymphokines, antibodies tosurface cancer proteins, and/or small molecular therapies likeVorinostat. Particular examples of immunotherapy agents that may be usedin conjunction with the compositions and methods described hereininclude anti-PD-1 antibodies and antigen-binding fragments thereof, suchas nivolumab, pembrolizumab, avelumab, durvalumab, and atezolizumab, aswell as anti-PD-L1 antibodies and antigen-binding fragments thereof,such as atezolizumab and avelumab, and anti-CTLA-4 antibodies andantigen-binding fragments thereof, such as ipilimumab or tremelimumab.

As used herein, the term “monoclonal antibody” refers to an antibodythat is derived from a single clone, including any eukaryotic,prokaryotic, or phage clone, and not the method by which it is produced.

As used herein, the term “multi-specific antibodies” refers toantibodies that exhibit affinity for more than one target antigen.Multi-specific antibodies can have structures similar to fullimmunoglobulin molecules and include Fc regions, for example IgG Fcregions. Such structures can include, but not limited to, IgG-Fv,IgG-(scFv)₂, DVD-Ig, (scFv)₂-(scFv)₂-Fc and (scFv)₂-Fc-(scFv)₂. In caseof IgG-(scFv)₂, the scFv can be attached to either the N-terminal or theC-terminal end of either the heavy chain or the light chain. Exemplarymulti-specific molecules that include Fc regions and into whichanti-TNFR2 antibodies or antigen-binding fragments thereof can beincorporated have been reviewed by Kontermann, 2012, mAbs 4(2):182-197,Yazaki et al, 2013, Protein Engineering, Design & Selection26(3):187-193, and Grote et al, 2012, in Proetzel & Ebersbach (eds.),Antibody Methods and Protocols, Methods in Molecular Biology vol. 901,chapter 16:247-263; incorporated herein by reference. In someembodiments, antibody fragments can be components of multi-specificmolecules without Fc regions, based on fragments of IgG or DVD or scFv.Exemplary multi-specific molecules that lack Fc regions and into whichantibodies or antibody fragments can be incorporated include scFv dimers(diabodies), trimers (triabodies) and tetramers (tetrabodies), Fabdimers (conjugates by adhesive polypeptide or protein domains) and Fabtrimers (chemically conjugated), are described by Hudson and Souriau,2003, Nature Medicine 9:129-134; incorporated herein by reference.

As used herein, the term “myeloid-derived suppressor cell” or “MDSC”refers to a cell of the immune system that modulates the activity of avariety of effector cells and antigen-presenting cells, such as T cells,NK cells, dendritic cells, and macrophages, among others. Myeloidderived suppressor cells are distinguished by their gene expressionprofile, and express all or a subset of proteins and small moleculesselected from the group consisting of B7-1 (CD80), B7-H1 (PD-L1), CCR2,CD1d, CD1d1, CD2, CD31 (PECAM-1), CD43, CD44, complement component C5aR1, F4/80 (EMR1), Fcγ RIII (CD16), Fcγ RII (CD32), Fcγ RIIA (CD32a), FcγRIIB (CD32b), Fcγ RIIB/C (CD32b/c), Fcγ RIIC (CD32c), Fcγ RIIIA (CD16A),Fcγ RIIIB (CD16b), galectin-3, GP130, Gr-1 (Ly-6G), ICAM-1 (CD54), IL-1RI, IL-4Rα, IL-6Rα, integrin α4 (CD49d), integrin αL (CD11a), integrinαM (CD11 b), M-CSFR, MGL1 (CD301a), MGL1/2 (CD301a/b), MGL2 (CD301b),nitric oxide, PSGL-1 (CD162), L-selectin (CD62L), siglec-3 (CD33),transferrin receptor (TfR), VEGFR1 (Flt-1), and VEGFR2 (KDR or Flk-1).Particularly, MDSCs do not express proteins selected from the groupconsisting of B7-2 (CD86), B7-H4, CD11c, CD14, CD21, CD23 (FccRII),CD34, CD35, CD40 (TNFRSF5), CD117 (c-kit), HLA-DR, and Sca-1 (Ly6).

As used herein, the terms “neutral TNFR2 polypeptide” and“phenotype-neutral TNFR2 polypeptide” refer to a polypeptide (such as asingle-chain polypeptide, an antibody, or an antibody fragment) thatbinds TNFR2 and does not exert an antagonistic or an agonistic effect onTNFR2 activation. For instance, a TNFR2 polypeptide is a neutral TNFR2polypeptide if the polypeptide binds TNFR2 and neither potentiates norsuppresses TNFR2 activation, for instance, as assessed by measuring theproliferation of TNFR2-expressing cells (e.g., T-reg cells, TNFR2+cancer cells, and/or MDSCs) and/or by measuring the expression of one ormore NFκB target genes, such as CHUK, NFKBIE, NFKBIA, MAP3K11, TRAF2,TRAF3, relB, and/or cIAP2/BIRC3.

As used herein, the term “non-native constant region” refers to anantibody constant region that is derived from a source that is differentfrom the antibody variable region or that is a human-generated syntheticpolypeptide having an amino sequence that is different from the nativeantibody constant region sequence. For instance, an antibody containinga non-native constant region may have a variable region derived from anon-human source (e.g., a mouse, rat, or rabbit) and a constant regionderived from a human source (e.g., a human antibody constant region), ora constant region derived from another primate, pig, goat, rabbit,hamster, cat, dog, guinea pig, member of the bovidae family (such ascattle, bison, buffalo, elk, and yaks, among others), cow, sheep, horse,or bison, among others).

As used herein, the term “percent (%) sequence identity” refers to thepercentage of amino acid (or nucleic acid) residues of a candidatesequence that are identical to the amino acid (or nucleic acid) residuesof a reference sequence after aligning the sequences and introducinggaps, if necessary, to achieve the maximum percent sequence identity(e.g., gaps can be introduced in one or both of the candidate andreference sequences for optimal alignment and non-homologous sequencescan be disregarded for comparison purposes). Alignment for purposes ofdetermining percent sequence identity can be achieved in various waysthat are within the skill in the art, for instance, using publiclyavailable computer software, such as BLAST, ALIGN, or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor measuring alignment, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For example, a reference sequence aligned for comparison with acandidate sequence may show that the candidate sequence exhibits from50% to 100% sequence identity across the full length of the candidatesequence or a selected portion of contiguous amino acid (or nucleicacid) residues of the candidate sequence. The length of the candidatesequence aligned for comparison purposes may be, for example, at least30%, (e.g., 30%, 40, 50%, 60%, 70%, 80%, 90%, or 100%) of the length ofthe reference sequence. When a position in the candidate sequence isoccupied by the same amino acid residue as the corresponding position inthe reference sequence, then the molecules are identical at thatposition.

As used herein, the term “primatized antibody” refers to an antibodycomprising framework regions from primate-derived antibodies and otherregions, such as CDRs and/or constant regions, from antibodies of anon-primate source. Methods for producing primatized antibodies areknown in the art. See e.g., U.S. Pat. Nos. 5,658,570; 5,681,722; and5,693,780; incorporated herein by reference. For instance, a primatizedantibody or antigen-binding fragment thereof described herein can beproduced by inserting the CDRs of a non-primate antibody orantigen-binding fragment thereof into an antibody or antigen-bindingfragment thereof that contains one or more framework regions of aprimate.

As used herein, the term “proliferation” in the context of a populationof cells, such as a population of TNFR2+ cells (e.g., T-reg cells,MDSCs, or TNFR2+ cancer cells) refers to mitotic and cytokineticdivision of a cell so as to produce a plurality of cells. Cellproliferation may be evidenced, for example, by a finding that thequantity of cells (e.g., TNFR2+ cells) in a sample of cells hasincreased over a given time period, such as over the course of one ormore hours, days, or weeks. One of skill in the art may monitor cellproliferation using a variety of known techniques, such as by way ofvisual microscopy, hemocytometry, flow cytometry, fluorescence activatedcell sorting, and other assays known in the art. In the presentdisclosure, cell proliferation is considered to be “inhibited” when therate of proliferation of a population of cells, such as a population ofTNFR2+ cells contacted with an antagonistic TNFR2 polypeptide describedherein, is decreased relative to the rate of proliferation of apopulation of control cells, such as a population of TNFR2+ cells notcontacted with the antagonistic TNFR2 polypeptide. A decrease in therate of proliferation may manifest, for example, as a reduction in thequantity of cells of interest in a sample over a given time period, suchas a reduction in the quantity of cells of interest in a sample of 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or more, over a given timeperiod. Additionally or alternatively, inhibition of cell proliferationmay be evidenced by a finding that the rate at which cells of interest(e.g., TNFR2+ cells contacted with an antagonistic TNFR2 polypeptidedescribed herein) are dividing is reduced, e.g., by %, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 60%, 70%, 80%, 90%, or more, relative to the rate at whichcontrol cells (e.g., TNFR2+ cells not contacted with the antagonisticTNFR2 polypeptide) are dividing.

As used herein, the term “operatively linked” in the context of apolynucleotide fragment is intended to mean that the two polynucleotidefragments are joined such that the amino acid sequences encoded by thetwo polynucleotide fragments remain in-frame.

As used herein, the term “pharmacokinetic profile” refers to theabsorption, distribution, metabolism, and clearance of a drug over timefollowing administration of the drug to a patient.

As used herein, a “recessive antagonist” of TNFR2 is an antagonist(e.g., an antagonistic polypeptide, such as a single-chain polypeptide,antibody, or antigen-binding fragment thereof) that inhibits TNFR2activation to a significantly lesser extent in the presence of a TNFR2agonist, such as TNFα, or IL-2 relative to the extent of inhibition ofthe same antagonist as measured in the absence of a TNFR2 agonist, suchas TNFα, or IL-2. For example, a TNFR2 antagonist is a recessiveantagonist if the IC₅₀ of the antagonist increases by, e.g., 10-fold,20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold,100-fold, or more in the presence of a TNFR2 agonist (e.g., TNFα orBacillus Calmette-Guérin (BCG)) or IL-2 relative to the IC₅₀ of theantagonist as measured in the same assay the absence of a TNFR2 agonist,such as TNFα, or IL-2. Inhibition of TNFR2 activation can be assessed,for instance, by measuring the inhibition of proliferation of TNFR2+cells, such as T-reg cells, cancer cells that express TNFR2, ormyeloid-derived suppressor cells, as well as by measuring the inhibitionof NFκB signaling (e.g., by monitoring the reduction in expression ofone or more genes selected from the group consisting of CHUK, NFKBIE,NFKBIA, MAP3K11, TRAF2, TRAF3, relB, and cIAP2/BIRC3 in a conventionalgene expression assay).

As used herein, the term “regulatory sequence” includes promoters,enhancers and other expression control elements (e.g., polyadenylationsignals) that control the transcription or translation of the antibodychain genes. Such regulatory sequences are described, for example, inGoeddel, Gene Expression Technology: Methods in Enzymology 185 (AcademicPress, San Diego, Calif., 1990); incorporated herein by reference.

As used herein, the term “scFv” refers to a single-chain Fv antibody inwhich the variable domains of the heavy chain and the light chain froman antibody have been joined to form one chain. scFv fragments contain asingle polypeptide chain that includes the variable region of anantibody light chain (VL) (e.g., CDR-L1, CDR-L2, and/or CDR-L3) and thevariable region of an antibody heavy chain (VH) (e.g., CDR-H1, CDR-H2,and/or CDR-H3) separated by a linker. The linker that joins the VL andVH regions of a scFv fragment can be a peptide linker composed ofproteinogenic amino acids. Alternative linkers can be used to so as toincrease the resistance of the scFv fragment to proteolytic degradation(e.g., linkers containing D-amino acids), in order to enhance thesolubility of the scFv fragment (e.g., hydrophilic linkers such aspolyethylene glycol-containing linkers or polypeptides containingrepeating glycine and serine residues), to improve the biophysicalstability of the molecule (e.g., a linker containing cysteine residuesthat form intramolecular or intermolecular disulfide bonds), or toattenuate the immunogenicity of the scFv fragment (e.g., linkerscontaining glycosylation sites). scFv molecules are known in the art andare described, e.g., in U.S. Pat. No. 5,892,019, Flo et al., (Gene77:51, 1989); Bird et al., (Science 242:423, 1988); Pantoliano et al.,(Biochemistry 30:10117, 1991); Milenic et al., (Cancer Research 51:6363,1991); and Takkinen et al., (Protein Engineering 4:837, 1991). The VLand VH domains of a scFv molecule can be derived from one or moreantibody molecules. It will also be understood by one of ordinary skillin the art that the variable regions of the scFv molecules describedherein can be modified such that they vary in amino acid sequence fromthe antibody molecule from which they were derived. For example, in oneembodiment, nucleotide or amino acid substitutions leading toconservative substitutions or changes at amino acid residues can be made(e.g., in CDR and/or framework residues). Alternatively or in addition,mutations are made to CDR amino acid residues to optimize antigenbinding using art recognized techniques. scFv fragments are described,for example, in WO 2011/084714; incorporated herein by reference.

As used herein, the phrase “specifically binds” refers to a bindingreaction which is determinative of the presence of an antigen in aheterogeneous population of proteins and other biological molecules thatis recognized, e.g., by an antibody or antigen-binding fragment thereof,with particularity. An antibody or antigen-binding fragment thereof thatspecifically binds to an antigen will bind to the antigen with a K_(D)of less than 100 nM. For example, an antibody or antigen-bindingfragment thereof that specifically binds to an antigen will bind to theantigen with a K_(D) of up to 100 nM (e.g., between 1 pM and 100 nM). Anantibody or antigen-binding fragment thereof that does not exhibitspecific binding to a particular antigen or epitope thereof will exhibita K_(D) of greater than 100 nM (e.g., greater than 500 nm, 1 μM, 100 μM,500 μM, or 1 mM) for that particular antigen or epitope thereof. Avariety of immunoassay formats may be used to select antibodiesspecifically immunoreactive with a particular protein or carbohydrate.For example, solid-phase ELISA immunoassays are routinely used to selectantibodies specifically immunoreactive with a protein or carbohydrate.See, Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring HarborPress, New York (1988) and Harlow & Lane, Using Antibodies, A LaboratoryManual, Cold Spring Harbor Press, New York (1999), for a description ofimmunoassay formats and conditions that can be used to determinespecific immunoreactivity.

As used herein, the terms “subject” and “patient” refer to an organismthat receives treatment for a particular disease or condition asdescribed herein (such as cancer or an infectious disease). Examples ofsubjects and patients include mammals, such as humans, primates, pigs,goats, rabbits, hamsters, cats, dogs, guinea pigs, members of thebovidae family (such as cattle, bison, buffalo, elk, and yaks, amongothers), cows, sheep, horses, and bison, among others, receivingtreatment for diseases or conditions, for example, cell proliferationdisorders, such as cancer or infectious diseases.

As used herein, the term “transfection” refers to any of a wide varietyof techniques commonly used for the introduction of exogenous DNA into aprokaryotic or eukaryotic host cell, e.g., electroporation, lipofection,calcium-phosphate precipitation, DEAE-dextran transfection and the like.

As used herein, the terms “treat” or “treatment” refer to therapeutictreatment, in which the object is to prevent or slow down (lessen) anundesired physiological change or disorder, such as the progression of acell proliferation disorder, such as cancer, or an infectious disease.Beneficial or desired clinical results include, but are not limited to,alleviation of symptoms, diminishment of extent of disease, stabilized(i.e., not worsening) state of disease, delay or slowing of diseaseprogression, amelioration or palliation of the disease state, andremission (whether partial or total), whether detectable orundetectable. Those in need of treatment include those already with thecondition or disorder, as well as those prone to have the condition ordisorder or those in which the condition or disorder is to be prevented.

As used herein, the term “tumor microenvironment” refers to cancer cellsthat form a tumor and the population of non-cancer cells, molecules,and/or blood vessels within the tumor or that border or surround thecancer cells.

As used herein, the terms “tumor necrosis factor receptor superfamily,”“TNFR superfamily,” or “TNFRS” refer to a group of type I transmembraneproteins with a carboxy-terminal intracellular domain and anamino-terminal extracellular domain characterized by a commoncysteine-rich domain (CRD). The TNFR superfamily includes receptors thatmediate cellular signaling as a consequence of binding to one or moreligands in the TNF superfamily. The TNFR superfamily can be divided intotwo subgroups: receptors containing the intracellular death domain andthose lacking this domain. The death domain is an 80 amino acid motifthat propagates apoptotic signal transduction cascades followingreceptor activation. Exemplary TNFR super family members that containthe intracellular death domain include TNFR1, while TNFR2 represents aTNFR super family protein that does not contain this domain. Members ofthe TNFR superfamily include TNFR1, TNFR2, RANK, CD30, CD40, Lymphotoxinbeta receptor (LT-βR), OX40, Fas receptor, Decoy receptor 3 (DCR3),CD27, 4-1 BB, Death receptor 4 (DR4), Death receptor 5 (DR5), Decoyreceptor 1 (DCR1), Decoy receptor 2 (DCR2), Osteoprotegerin, TWEAKreceptor, TACI, BAFF receptor, Herpesvirus entry mediator, Nerve growthfactor receptor, B cell maturation antigen, Glucocorticoid-inducedTNFR-related, TROY, Death receptor 6 (DR6), Death receptor 3 (DR3), andEctodysplasin A2 receptor.

As used herein, the terms “tumor necrosis factor receptor 2 signaling,”“TNFR2 signaling,” “TNFR2 signal transduction,” and the like, are usedinterchangeably and refer to the cellular events that normally occurupon activation of TNFR2 on the surface of a TNFR2+ cell, such as T-regcell, MDSC, or TNFR2+ cancer cell, by an endogenous TNFR2 ligand, suchas TNFα. TNFR2 signaling may be evidenced by a finding that expressionis increased for one or more genes selected from the group consisting ofCHUK, NFKBIE, NFKBIA, MAP3K11, TRAF2, TRAF3, relB, and cIAP2/BIRC3.TNFR2 signaling is considered to be “inhibited” as used herein when theexpression (and/or post-translational modification in the event thatsuch a modification is required for activity of the encoded protein) ofone or more, or all, of the foregoing genes is decreased in a TNFR2+cell upon contacting the cell with an agent, such as a TNFR2 antagonistpolypeptide described herein, relative to a TNFR2+ cell that is notcontacted with the agent (e.g., TNFR2 antagonist polypeptide). TNFR2signaling is considered to be “inhibited,” for example, when theexpression or post-translational modification (e.g., phosphorylation) ofone or more of CHUK, NFKBIE, NFKBIA, MAP3K11, TRAF2, TRAF3, relB, orcIAP2/BIRC3, in a TNFR2+ cell contacted with an antagonistic TNFR2polypeptide is decreased by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 100% relative to the expression orpost-translational modification (e.g., phosphorylation) of one or moreof these genes in a TNFR2+ cell not contacted with the antagonisticTNFR2 polypeptide. Exemplary assays that can be used to determineexpression level and phosphorylation state are known in the art andinclude, e.g., Western blot assays to determine protein content andquantitative reverse transcription polymerase chain reaction (RT-PCR)experiments to determine mRNA content.

As used herein the term “variable region CDR” includes amino acids in aCDR or complementarity determining region as identified using sequenceor structure based methods. As used herein, the term “CDR” or“complementarity determining region” refers to the noncontiguousantigen-binding sites found within the variable regions of both heavyand light chain polypeptides. These particular regions have beendescribed by Kabat et al., J. Biol. Chem. 252:6609-6616, 1977 and Kabat,et al., Sequences of Proteins of Immunological Interest, Fifth Edition,U.S. Department of Health and Human Services, NIH Publication No.91-3242, 1991; by Chothia et al., (J. Mol. Biol. 196:901-917, 1987), andby MacCallum et al., (J. Mol. Biol. 262:732-745, 1996) where thedefinitions include overlapping or subsets of amino acid residues whencompared against each other. The term “CDR” may be, for example, a CDRas defined by Kabat based on sequence comparisons.

As used herein, the term “vector” includes a nucleic acid vector, e.g.,a DNA vector, such as a plasmid, a RNA vector, virus or other suitablereplicon (e.g., viral vector). A variety of vectors have been developedfor the delivery of polynucleotides encoding exogenous proteins into aprokaryotic or eukaryotic cell. Examples of such expression vectors aredisclosed in, e.g., WO 1994/11026; incorporated herein by reference.Expression vectors described herein contain a polynucleotide sequence aswell as, e.g., additional sequence elements used for the expression ofproteins and/or the integration of these polynucleotide sequences intothe genome of a mammalian cell. Certain vectors that can be used for theexpression of antibodies and antibody fragments described herein includeplasmids that contain regulatory sequences, such as promoter andenhancer regions, which direct gene transcription. Other useful vectorsfor expression of antibodies and antibody fragments containpolynucleotide sequences that enhance the rate of translation of thesegenes or improve the stability or nuclear export of the mRNA thatresults from gene transcription. These sequence elements include, e.g.,5′ and 3′ untranslated regions, an internal ribosomal entry site (IRES),and polyadenylation signal site in order to direct efficienttranscription of the gene carried on the expression vector. Theexpression vectors described herein may also contain a polynucleotideencoding a marker for selection of cells that contain such a vector.Examples of a suitable marker include genes that encode resistance toantibiotics, such as ampicillin, chloramphenicol, kanamycin, ornourseothricin.

As used herein, the term “VH” refers to the variable region of animmunoglobulin heavy chain of an antibody, including the heavy chain ofan Fv, scFv, or Fab. References to “VL” refer to the variable region ofan immunoglobulin light chain, including the light chain of an Fv, scFv,dsFv or Fab. Antibodies (Abs) and immunoglobulins (Igs) areglycoproteins having the same structural characteristics. Whileantibodies exhibit binding specificity to a specific target,immunoglobulins include both antibodies and other antibody-likemolecules which lack target specificity. Native antibodies andimmunoglobulins are usually heterotetrameric glycoproteins of about150,000 Daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each heavy chain of a native antibody has atthe amino terminus a variable domain (VH) followed by a number ofconstant domains. Each light chain of a native antibody has a variabledomain at the amino terminus (VL) and a constant domain at the carboxyterminus.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the amino acid sequence of human TNFR2 (SEQ ID NO: 7).Human TNFR2 is numbered herein starting with an N-terminal methionine atposition 1 and concluding with a C-terminal serine at position 461 (SEQID NO: 7). All references to amino acid positions within TNFR2 are madein the context of the TNFR2 numbering scheme shown in FIG. 1 . Thebinding of residues shown in bold and underlined font (KCRPG, SEQ ID NO:19), along with other epitopes present in cysteine-rich domain 3 (CRD3)and CRD4 of human TNFR2 (residues 121-162 and 162-202 of SEQ ID NO: 7,respectively) and equivalent regions within TNFR2 of non-humans, such asnon-human mammals, promotes antagonism of TNFR2 signaling. The bindingof italicized residues (KCSPG, SEQ ID NO: 12), along with other epitopespresent in CRD1 of human TNFR2 (residues 48-76 of SEQ ID NO: 7) andequivalent regions within TNFR2 of non-humans, such as non-humanmammals, disfavors TNFR2 antagonism.

FIG. 2 is a graph contrasting the effects of murine monoclonal TNFR2antagonist antibody TNFRAB2 on the viability of regulatory T (T-reg)cells in vitro (left) with the effects of a human chimeric version ofTNFRAB2 on the viability of T-reg cells under the same assay conditions(right). TNFRAB2 is a murine IgG2 antibody, and the human chimeric TNFR2antagonist antibody tested in this example has a human IgG1 isotype.Values along the x-axis represent antibody concentration in units ofμg/ml. Values along the y-axis represent the percentage of T-reg cellspresent in an in vitro cell sample after the sample is incubated withthe indicated concentration of TNFR2 antibody.

FIG. 3 is a graph contrasting the effects of murine monoclonal TNFR2antagonist antibody TNFRAB2 on the quantity of effector T cells in an invitro sample (left) with the effects of a human chimeric version ofTNFRAB2 on the quantity of effector T cells in an in vitro sample underthe same assay conditions (right). The human chimeric TNFR2 antagonistantibody is the same one described in FIG. 2 . Values along the x-axisrepresent antibody concentration in units of μg/ml. Values along they-axis represent the percentage of T effector cells present in an invitro cell sample after the sample is incubated with the indicatedconcentration of TNFR2 antibody.

FIG. 4 is a graph contrasting the effects of murine monoclonal TNFR2antagonist antibody TNFRAB2 on the quantity of TNFR2+ SW480 colon cancercells in an in vitro sample (left) with the effects of a human chimericversion of TNFRAB2 on the quantity of TNFR2+ SW480 colon cancer cells inan in vitro sample under the same assay conditions (right). The humanchimeric TNFR2 antagonist antibody is the same one described in FIG. 2 .Values along the x-axis represent antibody concentration in units ofμg/ml. Values along the y-axis represent the quantity of TNFR2+ SW480colon cancer cells present in an in vitro cell sample after the sampleis incubated with the indicated concentration of TNFR2 antibody.

FIG. 5 is a graph contrasting the effects of murine monoclonal TNFR2antagonist antibody TNFRAB2 on the quantity of effector T cells in an invitro sample (left) with the effects of a human chimeric version ofTNFRAB2 on the quantity of effector T cells in an in vitro sample underthe same assay conditions (right). The human chimeric TNFR2 antagonistantibody is the same one described in FIG. 2 . Values along the x-axisrepresent antibody concentration in units of μg/ml. Values along they-axis represent the percentage of T effector cells present in an invitro cell sample after the sample is incubated with the indicatedconcentration of TNFR2 antibody.

FIG. 6 is a graph contrasting the effects of murine monoclonal TNFR2antagonist antibody TNFRAB2 on the viability of T-reg cells in vitro(left) with the effects of a human chimeric version of TNFRAB2 on theviability of T-reg cells under the same assay conditions (right). Thehuman chimeric TNFR2 antagonist antibody is the same one described inFIG. 2 . Values along the x-axis represent antibody concentration inunits of μg/ml. Values along the y-axis represent the percentage ofT-reg cells present in an in vitro cell sample after the sample isincubated with the indicated concentration of TNFR2 antibody.

FIG. 7 is a graph contrasting the effects of murine monoclonal TNFR2antagonist antibody TNFRAB2 on the quantity of TNFR2+ SW480 colon cancercells in an in vitro sample (left) with the effects of a human chimericversion of TNFRAB2 on the quantity of TNFR2+ SW480 colon cancer cells inan in vitro sample under the same assay conditions (right). The humanchimeric TNFR2 antagonist antibody is the same one described in FIG. 2 .Values along the x-axis represent antibody concentration in units ofμg/ml. Values along the y-axis represent the quantity of TNFR2+ SW480colon cancer cells present in an in vitro cell sample after the sampleis incubated with the indicated concentration of TNFR2 antibody.

FIG. 8 is a graph contrasting the effects of murine monoclonal TNFR2antagonist antibody TNFRAB2 on the quantity of effector T cells in an invitro sample (left) with the effects of a human chimeric version ofTNFRAB2 on the quantity of effector T cells in an in vitro sample underthe same assay conditions (right). The human chimeric TNFR2 antagonistantibody tested in this example has a human IgG2 isotype and has a humanIgG2 hinge region featuring C232S and C233S amino acid substitutions.Values along the x-axis represent antibody concentration in units ofμg/ml. Values along the y-axis represent the percentage of T effectorcells present in an in vitro cell sample after the sample is incubatedwith the indicated concentration of TNFR2 antibody.

FIG. 9 is a graph contrasting the effects of murine monoclonal TNFR2antagonist antibody TNFRAB2 on the viability of T-reg cells in vitro(left) with the effects of a human chimeric version of TNFRAB2 on theviability of T-reg cells under the same assay conditions (right). Thehuman chimeric TNFR2 antagonist antibody tested in this example has ahuman IgG2 isotype and has a human IgG2 hinge region featuring C232S andC233S amino acid substitutions. Values along the x-axis representantibody concentration in units of μg/ml. Values along the y-axisrepresent the percentage of T-reg cells present in an in vitro cellsample after the sample is incubated with the indicated concentration ofTNFR2 antibody.

FIG. 10 is a graph contrasting the effects of murine monoclonal TNFR2antagonist antibody TNFRAB2 on the quantity of TNFR2+ SW480 colon cancercells in an in vitro sample (left) with the effects of a human chimericversion of TNFRAB2 on the quantity of TNFR2+ SW480 colon cancer cells inan in vitro sample under the same assay conditions (right). The humanchimeric TNFR2 antagonist antibody tested in this example has a humanIgG2 isotype and has a human IgG2 hinge region featuring C232S and C233Samino acid substitutions. Values along the x-axis represent antibodyconcentration in units of μg/ml. Values along the y-axis represent thequantity of TNFR2+ SW480 colon cancer cells present in an in vitro cellsample after the sample is incubated with the indicated concentration ofTNFR2 antibody.

FIG. 11 is a graph demonstrating the TNFR2+ cancer cell-killingproperties of a chimeric variant of monoclonal antibody TNFRAB2. Thehuman chimeric TNFR2 antagonist antibody tested in this example has ahuman IgG2 isotype and has a human IgG2 hinge region featuring C232S andC233S amino acid substitutions. Values along the x-axis represent thenumber of days following the treatment of TNFR2+ SW480 tumor cells withthe chimeric TNFRAB2 variant antibody. Values along the y-axis representSW480 tumor volume, in units of cubic millimeters, following treatmentwith the TNFRAB2 variant antibody. Tumor volume values observedfollowing treatment with the TNFRAB2 variant antibody (squares) arecompared to values observed following treatment with vehicle control(circles).

FIG. 12 is an image showing the results of a polyacrylamide gelelectrophoresis separation of a chimeric TNFRAB2 variant antibodycontaining a human IgG2 constant domain, along with a wild-type humanIgG2 hinge region, and the variable domain of the murine TNFRAB2monoclonal antibody described herein. The gel electrophoresis separationshown in this figure was conducted under non-reducing conditions. Fourunique bands were observed upon performing this separation,corresponding to the IgG2-A, IgG2-B, IgG2-A/B₁, and IgG2-A/B₂disulfide-bonded isoforms of the human IgG2 isotype. For clarity, thesebands are highlighted in white boxes.

FIGS. 13A-13D are a series of schematics comparing the disulfide bondingarrangement present in each of the IgG2-A (FIG. 13A), IgG2-B (FIG. 13B),IgG2-A/B₁ (FIG. 13C), and IgG2-A/B₂ (FIG. 13D) isoforms of a human IgG2isotype antibody. Thin lines represent disulfide bonds connectingvarious portions of each antibody heavy chain or light chain, which arerepresented by shaded rectangles. Heavy chains are represented by thelonger, outermost rectangles of each antibody. Within each heavy chain,black shading denotes the constant region, and light shading denotes thevariable region. Light chains are represented by the shorter, innermostrectangles of each antibody. Within each light chain, darker shadingdenotes the constant region, and lighter shading denotes the variableregion.

DETAILED DESCRIPTION

Antagonistic TNFR2 polypeptides described herein, such as single-chainpolypeptides, antibodies, antigen-binding fragments thereof, andconstructs thereof, inhibit the activation of TNFR2 on TNFR2-expressingcells. This may be effectuated, for instance, by binding TNFR2 (e.g., onthe exterior surface of a T-reg cell, a cancer cell that expressesTNFR2, or a myeloid-derived suppressor cell (MDSC)) and preventing thereceptor from adopting a three-dimensional conformation that is suitablefor binding its cognate ligand, TNFα. TNFα potentiates TNFR2 signalingby nucleating a trimer of TNFR2 proteins. It is this trimerization eventthat brings individual TNFR2 proteins into close proximity and initiatesTNFR2 signaling via the MAPK/NFκB/TRAF2/3 pathway, which ultimatelyleads to cell growth and escape from apoptosis. Antagonistic TNFR2polypeptides described herein can antagonize this interaction, forinstance, by binding the receptor and preventing receptor trimerization.For instance, one mechanism by which this may occur is through theformation of an anti-parallel TNFR2 dimer, which is an inactivestructural form of the receptor.

The TNFR2 polypeptides described herein specifically bind to epitopeswithin TNFR2 that promote receptor antagonism and various advantageousdownstream biological activities. Human TNFR2 contains fourcysteine-rich domains (CRDs): CRD1 (amino acid residues 48-76 of SEQ IDNO: 7), CRD2 (amino acid residues 78-120 of SEQ ID NO: 7), CRD3 (aminoacid residues 121-162 of SEQ ID NO: 7), and CRD4 (amino acid residues162-202 of SEQ ID NO: 7). Antagonistic TNFR2 polypeptides describedherein specifically bind TNFR2 at one or more epitopes within CRD3and/or CRD4. In some embodiments, the antagonistic TNFR2 polypeptides donot bind epitopes within CRD1 and/or CRD2. For example, the polypeptides(e.g., single-chain polypeptides, antibodies, antigen-binding fragmentsthereof, or constructs thereof) of the disclosure may bind human TNFR2at an epitope within one or more of the following residues:

-   -   (a) amino acids 142-146 of SEQ ID NO: 7 (KCRPG);    -   (b) amino acids 142-149 of SEQ ID NO: 7 (KCRPGFGV);    -   (c) amino acids 137-144 of SEQ ID NO: 7 (CAPLRKCR);    -   (d) amino acids 150-190 of SEQ ID NO: 7        (RPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAI);    -   (e) amino acids 161-169 of SEQ ID NO: 7 (CKPCAPGTF);    -   (f) amino acids 75-128 of SEQ ID NO: 7        (CDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCAL),        optionally in which the epitope is within amino acids 80-86        (DSTYTQL), 91-98 (PECLSCGS), or 116-123 (RICTCRPG) of SEQ ID NO:        7;        -   (g) amino acids 174-184 (SSTDICRPHQI) of SEQ ID NO: 7;        -   (h) amino acids 126-140 (CALSKQEGCRLCAPL) of SEQ ID NO: 7;            and/or        -   (i) amino acids 156-165 (TSDVVCKPCA) of SEQ ID NO: 7;        -   or an equivalent epitope within TNFR2 of a non-human mammal,            such as a non-human mammal described herein.

The present disclosure is based, in part, on the discovery thatanti-TNFR2 polypeptides demonstrate substantially improved TNFR2antagonist effects when these molecules are in the form of an IgG2isotype. As described in the examples below, it has presently beendiscovered that this class of TNFR2 polypeptides exhibits a surprisinglysuperior ability to disrupt TNFR2 signaling, attenuate T-reg cell andcancer cell growth, and augment the proliferation of effector T cellsrelative to TNFR2-binding polypeptides of other isotypes.

Another discovery underlying the present disclosure is the finding thatantagonistic TNFR2 polypeptides that contain antigen-binding sitesspatially separated from one another by about 133 Å or more exhibitunexpectedly superior TNFR2 antagonist effects relative to polypeptidesthat specifically bind TNFR2 at one or more of the epitopes describedabove but that contain antigen-binding sites separated from one anotherby fewer than about 133 Å. Examples of such polypeptides include IgG1antibodies and antigen-binding fragments thereof that containantigen-binding sites separated from one another by about 117 Å and IgG3antibodies and antigen-binding fragments thereof that containantigen-binding sites separated from one another by 125 Å.

Antagonistic TNFR2 polypeptides of the disclosure can be formulated intopharmaceutical compositions. Preferably, the polypeptides present in thepharmaceutical composition adopt a single disulfide-bonded isoform. Forexample, pharmaceutical compositions of the disclosure include thosecontaining an antagonist TNFR2 polypeptide in which, e.g., 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%,99.99%, or more, of the polypeptide in the pharmaceutical composition ispresent in a single disulfide-bonded isoform. Antagonistic TNFR2polypeptides of the disclosure may advantageously adopt an IgG2-Adisulfide-bonded isoform, which has surprisingly been found to promote asubstantially more robust level of TNFR2 antagonism relative to otherIgG2 disulfide-bonded isoforms, such as the IgG2-B, IgG2-A/B₁, andIgG2-A/B₂ isoforms. These isoforms are shown graphically in FIGS.13A-13D. Polypeptides of the disclosure may be engineered topredominantly adopt an IgG2-A isoform, for example, by introducingmutations into the IgG2 hinge region that prohibit the formation ofother disulfide-bonded isoforms. Exemplary mutations in the amino acidsequence of a human IgG2 hinge region that promote the formation of theIgG2-A isoform at the exclusion of the remaining isoforms describedabove include the deletions and/or substitutions of the cysteineresidues at positions 232 and 233 of the wild-type human IgG2 hingeamino acid sequence, which is set forth in SEQ ID NO: 291. For example,to engineer an IgG2 antibody or antigen-binding fragment thereof so asto predominantly adopt the IgG2-A isoform, one may introduceconservative amino acid substitutions at cysteine residues 232 and/or233 of SEQ ID NO: 291. An exemplary IgG2 hinge region that existspredominantly in the IgG2-A isoform has the amino acid sequence of SEQID NO: 292, which contains C232S and C233S substitutions relative to SEQID NO: 291.

The following biological activities are examples of antagonistic TNFR2phenotypes that are exhibited by polypeptides of the disclosure to asuperior extent relative to TNFR2-binding polypeptides that (i) have anisotype other than IgG2, (ii) contain antigen-binding sites separatedfrom one another by fewer than 133 Å, and/or (iii) do not existpredominantly in a single disulfide-bonded isoform (e.g., the IgG2-Aisoform):

-   -   (a) Suppression of the proliferation of, and/or the direct        killing of, T-reg cells, for instance, by binding and        inactivating TNFR2 on the T-reg cell surface;    -   (b) Suppression of the proliferation of, and/or the direct        killing of, MDSCs, for instance, by binding and inactivating        TNFR2 on the MDSC surface;    -   (c) Promotion of the expansion of T effector cells, such as CD8+        T cells; and/or    -   (d) Suppression of the proliferation of, and/or the direct        killing of, TNFR2-expressing cancer cells, such as Hodgkin's        lymphoma cells, cutaneous non-Hodgkin's lymphoma cells, T cell        lymphoma cells, ovarian cancer cells, colon cancer cells,        multiple myeloma cells, renal cell carcinoma cells, skin cancer        cells, lung cancer cells, liver cancer cells, endometrial cancer        cells, hematopoietic or lymphoid cancer cells, central nervous        system cancer cells, breast cancer cells, pancreatic cancer        cells, stomach cancer cells, esophageal cancer cells, and upper        gastrointestinal cancer cells.

The sections that follow provide a description of exemplarycharacteristics of antagonistic TNFR2 polypeptides described herein,such as single-chain polypeptides, antibodies, antigen-binding fragmentsthereof, and constructs thereof, as well as their use in therapeuticmethods.

Antagonistic TNFR2 Polypeptides IgG2 Isotype Antibodies Promotes OptimalTNFR2 Antagonism

As described above and herein, optimal TNFR2 antagonism among human,humanized, and chimeric TNFR2 antagonist antibodies and antigen-bindingfragment thereof is achieved when the antibody or antibody fragment hasa human IgG2 isotype, particularly when the antibody or antibodyfragment has an IgG2-A disulfide-bonded isoform. The disulfide bondingpattern of the various isoforms of human IgG2 antibodies are shown inFIGS. 13A-13D. As shown in FIG. 13A, the IgG2-A isoform exhibitsdisulfide bonding between cysteine residues C133 of the heavy chain andC214 of the light chain, as well as disulfide bonds betweencorresponding cysteine residues C221, C222, C225, and C228 present oneach heavy chain.

To stabilize the IgG2-A disulfide-bonded isoform, mutations can beintroduced into the IgG2 hinge region so as to prevent, or reduce theoccurrence of, disulfide bonding between cysteine residues that arepresent as nonbonded thiols in the IgG2-A isoform. Examples of suchmutations are amino acid substitutions or deletions at residues C232 andC233 of the human IgG2 hinge region. By removing one or both of theseresidues and optionally replacing these residues with amino acids thatare incapable of forming disulfide bonds, one can bias the disulfidebonding pattern in a population of IgG2 isoforms towards the IgG2-Aisoform. Examples of amino acid substitutions that can be used to obtaina population of IgG2-A isoform antibodies include conservative aminoacid substitutions, such as the C232S and C233S amino acidsubstitutions. Due to the similar molecular volume and polarity ofcysteine and serine, the C232S and C233S substitutions feature thebeneficial effect of preserving the steric and electronegativityproperties of the naturally-occurring cysteine residue while prohibitingthe formation of a disulfide bond at position 232 and/or 233 of the IgG2hinge region. By incorporating C232S and/or C233S substitutions into aTNFR2 antibody or fragment thereof, a population of TNFR2 antagonistantibodies or fragments having an IgG2-A isoform can be obtained.Methods of effectuating amino acid substitutions and deletions into anantibody or antigen-binding fragment thereof include mutagenesistechniques described herein and known in the art.

Spacing Between Antigen-Binding Sites

Antagonist TNFR2 polypeptide (e.g., single-chain polypeptides, antibody,antigen-binding fragment thereof, or construct thereof) described hereinmay contain antigen-binding sites (i.e., antigen-binding arms) that areseparated from one another by a distance of at least about 133 Å, whichis the spacing observed between antigen-binding arms in human IgG2isotype antibodies. As described in the examples below, it has beendiscovered that this spacing gives rise to antibodies having optimalTNFR2 antagonistic properties. TNFR2 antagonist polypeptides of thedisclosure include those containing antigen-binding arms separated by,e.g., a distance of from about 133 Å to about 160 Å, such as a distanceof about 133 Å, 134 Å, 135 Å, 136 Å, 137 Å, 138 Å, 139 Å, 140 Å, 141 Å,142 Å, 143 Å, 144 Å, 145 Å, 146 Å, 147 Å, 148 Å, 149 Å, 150 Å, 151 Å,152 Å, 153 Å, 154 Å, 155 Å, 156 Å, 157 Å, 158 Å, 159 Å, or 160 Å). Forexample, the polypeptide (e.g., a single-chain polypeptide, antibody,antigen-binding fragment thereof, or construct thereof) may containantigen-binding sites that are separated from one another by a distanceof from about 133 Å to about 150 Å, such as by a distance of about 133Å, 134 Å, 135 Å, 136 Å, 137 Å, 138 Å, 139 Å, 140 Å, 141 Å, 142 Å, 143 Å,144 Å, 145 Å, 146 Å, 147 Å, 148 Å, 149 Å, or 150 Å. In some embodiments,the antigen-binding are separated from one another by a distance of fromabout 133 Å to about 145 Å, such as by a distance of about 133 Å, 134 Å,135 Å, 136 Å, 137 Å, 138 Å, 139 Å, 140 Å, 141 Å, 142 Å, 143 Å, 144 Å, or145 Å. In some embodiments, the antigen-binding are separated from oneanother by a distance of from about 133 Å to about 139 Å, such as by adistance of about 133 Å, 134 Å, 135 Å, 136 Å, 137 Å, 138 Å, or 139 Å. Insome embodiments, the antigen-binding are separated from one another bya distance of from about 134 Å to about 139 Å, such as by a distance ofabout 134 Å, 135 Å, 136 Å, 137 Å, 138 Å, or 139 Å.

The TNFR2 antagonist polypeptides described herein may have, e.g., two,three, four, five, or more, antigen-binding arms separated by a distancespecified above. Examples of antibody fragments that have two or moreantigen-binding arms include, without limitation, diabodies, triabodies,F(ab′)₂ molecules, and tandem scFv (taFv) molecules, among others.Methods of generating these antibody fragments include peptide synthesisand recombinant protein expression techniques described herein and knownin the art.

There exist a variety of methods for measuring the distance betweenantigen-binding arms of an antibody or antibody fragment. For example,distances between antigen-binding arms of an antibody can be made byanalyzing the three-dimensional structure of an antibody or antibodyfragment using computer software, such as through the use of PYMOL® andother molecular imaging software. Three-dimensional structures ofpolypeptides, such as antibodies and antibody fragments, can becalculated using the data obtained from X-ray crystallographyexperiments and nuclear magnetic resonance (NMR) techniques known in theart. Examples of X-ray crystallography and NMR methods that can be usedto obtain three-dimensional polypeptide structures are described, e.g.,in Eigenbrot et al., Journal of Molecular Biology, 229:969-995, 1993;and Huang et al., Science, 317:1930-1934, 2007, the disclosures of eachof which are incorporated herein by reference in their entirety.

Uniformity of Populations of TNFR2 Antagonist Polypeptides

Pharmaceutical compositions can be generated in which the TNFR2antagonist polypeptide (e.g., antibody, antigen-binding fragmentthereof, single-chain polypeptide, or construct thereof) describedherein is present as a single disulfide-bonded isoform. For example, atleast 10%, or more, of the polypeptide in the pharmaceutical compositionmay be present as a single disulfide-bonded isoform (e.g., the IgG2-Aisoform). This may be achieved, for example, by way of amino acidsubstitutions or deletions at one or both of cysteine residues 232 and233 of the wild-type human IgG2 hinge region, thereby preventing orreducing the occurrence of disulfide bonding that could give rise to anIgG2 isoform other than IgG2-A (see, e.g., FIGS. 13A-13D). Thepharmaceutical compositions of the disclosure include those in which,for example, about 10% to about 99.999% of the antagonist TNFR2polypeptide in the pharmaceutical composition is present in a singledisulfide-bonded isoform, such as the IgG2-A isoform. For example,pharmaceutical compositions of the disclosure include those containingan antagonist TNFR2 polypeptide in which, e.g., 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, or more, ofthe polypeptide in the pharmaceutical composition is present in a singledisulfide-bonded isoform.

Techniques for measuring the relative quantities of variousdisulfide-bonded isoforms present in a sample of an antagonist TNFR2polypeptide include liquid chromatography techniques known in the artand described herein, such as those exemplified in Wypych et al., TheJournal of Biological Chemistry, 283:16194-16205, 2008, the disclosureof which is incorporated herein by reference in its entirety.

Effects on TNFR2/MAPK/TRAF2/3 Signal Transduction Cascades

Anti-TFNR2 polypeptides (e.g., single-chain polypeptides, antibodies,and antigen-binding fragments thereof) described herein are capable ofinteracting with and inhibiting the activity of TNFR2. Thus, theanti-TNFR2 polypeptides described herein can selectively antagonize theTNFα-TNFR2 interaction rather than promote TNFR2 signaling. This isparticularly important for therapeutic applications, such as cancerimmunotherapy, as TNFR2 activation upon association with TNFα leads topropagation of the MAPK and TRAF2/3 signal cascade and activation ofNFκB-mediated transcription of genes involved in T-reg cell growth andescape from apoptosis (Faustman, et al., Nat. Rev. Drug Disc.,9:482-493, 2010). The TNFR2 polypeptides (e.g., single-chainpolypeptides, antibodies, and antigen-binding fragments thereof)described herein bind TNFR2 at one or more specific epitopes thatprevent the receptor from forming a trimer with neighboring TNFR2proteins. This trimerization activates intracellular signaling by TNFR2,which, e.g., promotes proliferation of TNFR2+ cells, such as T-regcells, MDSCs, and/or TNFR2+ cancer cells. Advantageously, the TNFR2antagonist polypeptides described herein bind TNFR2 at particularepitopes so as to stabilize TNFR2 in an anti-parallel dimerconformation, in which TNFα binding sites are sterically inaccessible.This prevents TNFα from nucleating TNFR2 trimer formation, which wouldotherwise trigger TNFR2 signal transduction. The polypeptides describedherein can therefore be used to suppress the growth and proliferation ofTNFR2+ cells, such as T-reg cells, MDSCs, and TNFR2+ cancer cells. Thesuppression of T-reg and MDSC proliferation, for instance, enables theproliferation of T effector cells that can mount an immune responseagainst, e.g., a cancer cell or foreign pathogen. Thus, antagonisticTNFR2 polypeptides described herein can be administered to a mammaliansubject, such as a human, with a cell proliferation disorder or aninfectious disease, in order to enhance the effectiveness of an immuneresponse (e.g., an immune response against cancer cells or pathogenicorganisms) in the subject.

Effects on T-Reg Cell Proliferation

Antagonistic TNFR2 polypeptides, such as single-chain polypeptides,antibodies, or antigen-binding fragments thereof described herein, canbe used to attenuate the activity of T-reg cells that typicallyaccompanies T cell-mediated cytotoxicity against self cells, such as theattack of a tumor cell by a T lymphocyte. This can be achieved, forinstance, due to the ability of antagonistic TNFR2 polypeptidesdescribed herein to inhibit the proliferation of, and/or to directlykill, T-reg cells. Antagonistic TNFR2 polypeptides can, thus, beadministered (e.g., by any of a variety of routes of administrationdescribed herein) to a mammalian subject, such as a human, in order toprolong the duration of an adaptive immune response, such as a responseagainst a cancer cell or a pathogenic organism. In this way, forexample, antagonistic TNFR2 polypeptides, such as single-chainpolypeptides, antibodies, or antigen-binding fragments thereof describedherein, may synergize with existing techniques to enhance Tlymphocyte-based therapy for cancer and for infectious diseases. Forinstance, TNFR2 antagonists described herein may be administered tosuppress T-reg cell activity, thereby enhancing the cytotoxic effect oftumor reactive T cells. TNFR2 antagonists may also synergize withexisting strategies to promote tumor-reactive T cell survival, such aslymphodepletion and growth factor therapy, and in turn prolong theduration of anti-tumor reactivity in vivo.

Antagonistic TNFR2 polypeptides, such as single-chain polypeptides,antibodies, and antigen-binding fragments thereof can also be used totreat a broad array of infectious diseases in a mammalian subject (e.g.,a human), as inhibition of T-reg proliferation promotes the activity ofCD8+T lymphocytes capable of mounting an attack on pathogenic organisms.Additionally, antagonistic TNFR2 antibodies and antigen-bindingfragments thereof described herein can be used to treat a wide varietyof infectious diseases, such as Mycobacterium tuberculosis, in a humanor an agricultural farm animal (e.g., a bovine mammal, pig, cow, horse,sheep, goat, cat, dog, rabbit, hamster, guinea pig, or other non-humanmammal).

Direct Effects on TNFR2+ Cancer Cells

Antagonistic TNFR2 polypeptides, such as single-chain polypeptides,antibodies, or antigen-binding fragments thereof described herein maybind and inactivate TNFR2 on the surface of a cancer cell, such as aTNFR2+ tumor cell. For instance, antagonistic TNFR2 antibodies andantigen-binding fragments thereof described herein may bind TNFR2 on thesurface a T cell lymphoma cell (e.g., a Hodgkin's or cutaneousnon-Hodgkin's lymphoma cell), ovarian cancer cell, colon cancer cell,multiple myeloma cell, or renal cell carcinoma cell, among others. Theability of antagonistic TNFR2 antibodies and antigen-binding fragmentsthereof described herein to bind TNFR2 directly on a cancer cellprovides another pathway by which these molecules may attenuate cancercell survival and proliferation. For instance, an antagonistic TNFR2polypeptide described herein, such as an antagonistic TNFR2 single-chainpolypeptide, antibody, antigen-binding fragment thereof, or construct,may bind TNFR2 directly on the surface of a cancer cell (e.g., acutaneous T cell lymphoma cell, ovarian cancer cell, colon cancer cell,or multiple myeloma cell, such as an ovarian cancer cell) in order tosuppress the ability of the cell to proliferate and/or to promoteapoptosis of the cell.

TNFR2 Antagonist Polypeptides are not Reliant on AdditionalTNFR2-Binding Agents for Activity

Significantly, antagonistic TNFR2 polypeptides, such as single-chainpolypeptides, antibodies, or antigen-binding fragments thereof describedherein, are capable of binding TNFR2 and suppressing TNFR2-mediatedsignaling without the need for an endogenous TNFR2-binding agent, suchas TNFα. Antagonistic TNFR2 polypeptides, such as single-chainpolypeptides, antibodies, and antigen-binding fragments thereofdescribed herein do not require TNFα to attenuate T-reg and/or cancercell proliferation. Without being limited by mechanism, antagonisticTNFR2 antibodies or antigen-binding fragments thereof described hereinmay exhibit this property due to the ability of these antibodies orantigen-binding fragments thereof to bind TNFR2 at particular epitopesthat, when bound, stabilize the anti-parallel dimer conformation of thisreceptor. This structural configuration is not capable of potentiatingNFκB signaling. By maintaining TNFR2 in an inactive structural state,antagonistic TNFR2 polypeptides described herein may prevent TNFR2agonists from restoring cell growth and/or may result in the directkilling (e.g., by apoptosis) of a TNFR2+ cell, such as a T-reg cell,MDSC, or TNFR2+ cancer cell).

For instance, antagonistic TNFR2 polypeptides, such as single-chainpolypeptides, antibodies, antigen-binding fragments thereof, andconstructs thereof described herein, may bind TNFR2 on the surface of aTNFR2+ cell, such as a T-reg cell, cancer cell, or myeloid-derivedsuppressor cell (MDSC) and inhibit the proliferation of such cells inthe presence or absence of TNFα. For example, antagonistic TNFR2polypeptides, such as single-chain polypeptides, antibodies, andantigen-binding fragments thereof described herein, may inhibit theproliferation of such cells by, e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 100%, or more, relative to such cells that arenot treated with the TNFR2 antagonist polypeptide. The antagonisticTNFR2 polypeptide (e.g., single-chain polypeptide, antibody, orantigen-biding fragment thereof) may exhibit an IC₅₀ value in such acell proliferation assay that is largely unchanged by the presence orabsence of TNFα (e.g., an IC₅₀ value in the presence of TNFα that ischanged by less than 50%, 45%, 40%, 35%, 25%, 20%, 15%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, or less than 1% relative to the IC₅₀ value of theantagonistic TNFR2 polypeptide (e.g., single-chain polypeptide,antibody, or antigen-binding fragment thereof) in the same cellproliferation assay in the absence of TNFα). Examples of cell deathassays that can be used to measure the antagonistic effects of TNFR2antibodies are described herein, e.g., in Example 2 below. Similarly,antagonistic TNFR2 polypeptides, such as single-chain polypeptides,antibodies, antigen-binding fragments thereof, and constructs thereofdescribed herein, may inhibit TNFR2 signaling as assessed by measuringthe expression of one or more genes selected from the group consistingof CHUK, NFKBIE, NFKBIA, MAP3K11, TRAF2, TRAF3, relB, and cIAP2/BIRC3by, e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%,or more, relative to such cells that are not treated with the TNFR2antagonist polypeptide. The antagonistic TNFR2 polypeptide (e.g.,single-chain polypeptide, antibody, or antigen-biding fragment thereof)may exhibit an IC₅₀ value in such a gene expression assay that islargely unchanged by the presence or absence of TNFα (e.g., an IC₅₀value in the presence of TNFα that is changed by less than 50%, 45%,40%, 35%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or lessthan 1% relative to the IC₅₀ value of the antagonistic TNFR2 polypeptide(e.g., single-chain polypeptide, antibody, or antigen-binding fragmentthereof) in the same gene expression assay in the absence of TNFα).

Direct Killing of T-Reg Cells, MDSCs, and TNFR2+ Cancer Cells

Antagonistic TNFR2 polypeptides disclosed herein, such as single-chainpolypeptides, antibodies, antigen-binding fragments thereof, andconstructs thereof, may, for instance, not only reduce the proliferationof T-reg cells, TNFR2+ cancer cells, and/or MDSCs, but may also inducethe death of T-reg cells, TNFR2+ cancer cells, and/or MDSCs within asample (e.g., within a patient, such as a human patient). AntagonisticTNFR2 polypeptides described herein may be capable, for instance, ofreducing the total quantity of T-reg cells, cancer cells (such ascutaneous T cell lymphoma cells, ovarian cancer cells, colon cancercells, renal cell carcinoma cells or multiple myeloma cells, amongothers), and/or MDSCs in a sample treated with an antagonist TNFR2antibody or antigen-binding fragment thereof (such as a sample isolatedfrom a human patient undergoing treatment for cancer or an infectiousdisease as described herein) by, e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 100%, or more, relative to a sample not treatedwith an antagonist TNFR2 antibody or antigen-binding fragment thereof.

The ability of antagonistic TNFR2 polypeptides (e.g., single-chainpolypeptides, antibodies, and antigen-binding fragments) describedherein to attenuate T-reg, MDSC, and/or cancer cell growth may be due,in part, to the ability of these polypeptides to diminish the quantityof soluble TNFR2 within a sample (e.g., a sample isolated from a humanpatient undergoing treatment for cancer or an infectious disease asdescribed herein). In the absence of this beneficial activity, solubleTNFR2 can be secreted by, e.g., T-reg cells, and could otherwiseinterfere with the ability of TNFR2 antagonists to localize to TNFR2 atthe surface of a T-reg cell, TNFR2+ cancer cell, or MDSC by binding andsequestering such antagonists in the extracellular environment. Byreducing TNFR2 secretion, antagonistic TNFR2 antibodies orantigen-binding fragments thereof described herein may render T-regcells, TNFR2+ cancer cells, and/or MDSCs increasingly susceptible totherapeutic molecules, such as an antagonistic TNFR2 antibody orantigen-binding fragment thereof, and/or additional anti-cancer agents,such as those described herein or known in the art, that may be used inconjunction with the compositions and methods described herein.

Selective Modulation of Active (CD25^(Hi) and CD45RA^(Low)) T-Reg Cells

Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides,antibodies, antigen-binding fragments, and constructs thereof) describedherein may be capable of inhibiting the proliferation or reducing thetotal quantity of T-reg cells in a sample (e.g., a sample isolated froma human patient undergoing treatment for cancer or an infectious diseaseas described herein) and may act selectively on T-reg cells in anactively-dividing state. Antagonistic TNFR2 antibodies orantigen-binding fragments thereof described herein may selectivelytarget active T-reg cells that express CD25^(Hi) and CD45RA^(Low), e.g.,over resting T-reg cells that express CD25^(Med) and CD45RA^(Hi). Forinstance, antagonistic TNFR2 antibodies or antigen-binding fragmentsthereof described herein may be capable of reducing the proliferation ofT-reg cells expressing CD25^(Hi) and CD45RA^(Low) by, e.g., 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more relative toT-reg cells that do not express the CD25^(Hi) and CD45RA^(Low) proteins,such as T-reg cells that express CD25^(Med) and CD45RA^(Hi) proteins.

Modulation of T-Reg Cells, MDSCs, and T Effector Cells in the TumorMicroenvironment

Antagonist TNFR2 polypeptides described herein, such as single-chainpolypeptides, antibodies, and antigen-binding fragments thereof, mayinhibit the proliferation of T-reg cells with a greater potency in apatient suffering from cancer relative to a subject that does not havecancer. The antagonist TNFR2 polypeptides described herein, such assingle-chain polypeptides, antibodies, and antigen-binding fragmentsthereof, may inhibit the proliferation of T-reg cells with a greaterpotency in the microenvironment of a tumor relative to a site that isfree of cancer cells, such as a site distal from a tumor in a patientsuffering from cancer or in a subject without cancer. This effect may bedetermined using, for example, a cell death assay as described herein.For instance, the polypeptides described herein, such as single-chainpolypeptides, antibodies, antigen-binding fragments thereof, andconstructs thereof, may exhibit an IC₅₀ for reducing or inhibiting theproliferation of T-reg cells in the microenvironment of a tumor that isless than the IC₅₀ of the polypeptides for reducing or inhibiting theproliferation of T-reg cells in a site that is free of cancer cells by,for example, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold,1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold,35-fold, 40-fold, 45-fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold,or more. Examples of cell death assays that can be used to measure theantagonistic effects of anti-TNFR2 polypeptides are described herein,e.g., in Example 2, below. The polypeptides described herein, such assingle-chain polypeptides, antibodies, antigen-binding fragmentsthereof, and constructs thereof, may inhibit the proliferation of T-regcells or may promote the apoptosis of T-reg cells with a potency that isgreater in the microenvironment of a tumor containing TNFR2+ cancercells, such as Hodgkin's lymphoma cells, cutaneous non-Hodgkin'slymphoma cells, T cell lymphoma cells, ovarian cancer cells, coloncancer cells, multiple myeloma cells, renal cell carcinoma cells, skincancer cells, lung cancer cells, liver cancer cells, endometrial cancercells, hematopoietic or lymphoid cancer cells, central nervous systemcancer cells, breast cancer cells, pancreatic cancer cells, stomachcancer cells, esophageal cancer cells, and upper gastrointestinal cancercells, than in a site that is free of such cancer cells, such as a sitedistal from a tumor in a patient suffering from one or more of theforegoing cancers or a in a subject without cancer.

Additionally, or alternatively, the polypeptides described herein, suchas single-chain polypeptides, antibodies, antigen-binding fragmentsthereof, and constructs thereof, may inhibit the proliferation of MDSCswith a greater potency in a patient suffering from cancer relative to asubject that does not have cancer. The polypeptides described herein,such as single-chain polypeptides, antibodies, antigen-binding fragmentsthereof, and constructs thereof, may inhibit the proliferation of MDSCswith a greater potency in the microenvironment of a tumor relative to asite that is free of cancer cells, such as a site distal from a tumor ina patient suffering from cancer or in a subject without cancer. Thiseffect may be determined using, for example, a cell death assaydescribed herein. For instance, the polypeptides described herein, suchas single-chain polypeptides, antibodies, antigen-binding fragmentsthereof, and constructs thereof, may have an IC₅₀ for reducing orinhibiting the proliferation of MDSCs in the microenvironment of a tumorthat is less than the IC₅₀ of the polypeptides for reducing orinhibiting the proliferation of MDSCs in a site that is free of cancercells by, for example, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold,1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold,6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold,30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 100-fold, 1,000-fold,10,000-fold, or more. Examples of cell death assays that can be used tomeasure the antagonistic effects of anti-TNFR2 polypeptides aredescribed herein, e.g., in Example 2, below. The polypeptides describedherein, such as single-chain polypeptides, antibodies, antigen-bindingfragments thereof, and constructs thereof, may inhibit the proliferationof MDSCs or may promote the apoptosis of MDSCs with a potency that isgreater in the microenvironment of a tumor containing TNFR2+ cancercells, such as Hodgkin's lymphoma cells, cutaneous non-Hodgkin'slymphoma cells, T cell lymphoma cells, ovarian cancer cells, coloncancer cells, multiple myeloma cells, renal cell carcinoma cells, skincancer cells, lung cancer cells, liver cancer cells, endometrial cancercells, hematopoietic or lymphoid cancer cells, central nervous systemcancer cells, breast cancer cells, pancreatic cancer cells, stomachcancer cells, esophageal cancer cells, and upper gastrointestinal cancercells, than in a site that is free of such cancer cells, such as a sitedistal from a tumor in a patient suffering from one or more of theforegoing cancers or a in a subject without cancer.

Additionally, or alternatively, the polypeptides described herein, suchas single-chain polypeptides, antibodies, antigen-binding fragmentsthereof, and constructs thereof, may expand T effector cells, such asCD8+ cytotoxic T cells, with a greater potency in a patient sufferingfrom cancer relative to a subject that does not have cancer. In someembodiments, the polypeptides described herein, such as single-chainpolypeptides, antibodies, and antigen-binding fragments thereof, expandT effector cells, such as CD8+ cytotoxic T cells, with a greater potencyin the microenvironment of a tumor relative to a site that is free ofcancer cells, such as a site distal from a tumor in a patient sufferingfrom cancer or a in a subject without cancer. This effect may bedetermined using, for example, a cell proliferation assay describedherein. For instance, the polypeptides described herein, such assingle-chain polypeptides, antibodies, antigen-binding fragmentsthereof, and constructs thereof, may have an EC₅₀ for the expansion of Teffector cells in the microenvironment of a tumor that is less than theEC₅₀ of the polypeptides for expanding T effector cells in a site thatis free of cancer cells by, for example, 1.1-fold, 1.2-fold, 1.3-fold,1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold,3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold,100-fold, 1,000-fold, 10,000-fold, or more. Examples of cellproliferation assays that can be used to measure the effects ofanti-TNFR2 polypeptides on T effector cells are described herein, e.g.,in Example 2, below. The polypeptides described herein, such assingle-chain polypeptides, antibodies, antigen-binding fragmentsthereof, and constructs thereof, may directly expand T effector cells,such as CD8+ cytotoxic T cells, with a potency that is greater in themicroenvironment of a tumor containing TNFR2+ cancer cells, such asHodgkin's lymphoma cells, cutaneous non-Hodgkin's lymphoma cells, T celllymphoma cells, ovarian cancer cells, colon cancer cells, multiplemyeloma cells, renal cell carcinoma cells, skin cancer cells, lungcancer cells, liver cancer cells, endometrial cancer cells,hematopoietic or lymphoid cancer cells, central nervous system cancercells, breast cancer cells, pancreatic cancer cells, stomach cancercells, esophageal cancer cells, and upper gastrointestinal cancer cells,than in a site that is free of such cancer cells, such as a site distalfrom a tumor in a patient suffering from one or more of the foregoingcancers or a in a subject without cancer. The T effector cells (e.g.,CD8+ cytotoxic T cells) may, for example, specifically react with anantigen present on one or more cancer cells, such as Hodgkin's lymphomacells, cutaneous non-Hodgkin's lymphoma cells, T cell lymphoma cells,ovarian cancer cells, colon cancer cells, multiple myeloma cells, renalcell carcinoma cells, skin cancer cells, lung cancer cells, liver cancercells, endometrial cancer cells, hematopoietic or lymphoid cancer cells,central nervous system cancer cells, breast cancer cells, pancreaticcancer cells, stomach cancer cells, esophageal cancer cells, and uppergastrointestinal cancer cells, among cells of other cancers describedherein.

Activity of Antigen-Binding Fragments of Full-Length TNFR2 AntagonistAntibodies

Antagonistic TNFR2 antibodies described herein may inhibit, e.g., T-reg,cancer cell, and/or MDSC growth, or promote T effector cell growth, witha similar potency as that exhibited by antigen-binding fragments of suchantibodies. For instance, removal of the Fc region of an antagonisticTNFR2 antibody described herein may not alter the ability of themolecule to attenuate the proliferation or reduce the total quantity ofT-reg cells, MDSCs, and/or cancer cells in a sample (e.g., a sampleisolated from a human patient undergoing treatment for cancer or aninfectious disease as described herein). Antagonistic TNFR2 antibodiesand antigen-binding fragments thereof described herein may function, forinstance, by a pathway distinct from antibody-dependent cellularcytotoxicity (ADCC), in which a Fc region is required to recruiteffector proteins in order to induce cell death. Additionally,antagonistic TNFR2 antibodies or antigen-binding fragments thereof mayexhibit therapeutic activity in a variety of forms, such as asingle-chain polypeptide (e.g., a single-chain polypeptide containingone or more CDRs covalently bound to one another, for instance, by anamide bond, a thioether bond, a carbon-carbon bond, or a disulfidebridge), a monoclonal antibody or antigen-binding fragment thereof, apolyclonal antibody or antigen-binding fragment thereof, a humanizedantibody or antigen-binding fragment thereof, a primatized antibody orantigen-binding fragment thereof, a bispecific antibody orantigen-binding fragment thereof, a multi-specific antibody orantigen-binding fragment thereof, a dual-variable immunoglobulin domain,a monovalent antibody or antigen-binding fragment thereof, a chimericantibody or antigen-binding fragment thereof, a single-chain Fv molecule(scFv), a diabody, a triabody, a nanobody, an antibody-like proteinscaffold, a domain antibody, a Fv fragment, a Fab fragment, a F(ab′)2molecule, and a tandem scFv (taFv).

Specific Binding Properties of Antagonistic TNFR2 Polypeptides

The specific binding of a polypeptide, such as a single-chainpolypeptide, antibody, or antibody fragment described herein, to humanTNFR2 can be determined by any of a variety of established methods. Theaffinity can be represented quantitatively by various measurements,including the concentration of antibody needed to achieve half-maximalinhibition of the TNFα-TNFR2 interaction in vitro (IC₅₀) and theequilibrium constant (K_(D)) of the antibody-TNFR2 complex dissociation.The equilibrium constant, K_(D), that describes the interaction of TNFR2with an antibody described herein is the chemical equilibrium constantfor the dissociation reaction of a TNFR2-antibody complex intosolvent-separated TNFR2 and antibody molecules that do not interact withone another.

Polypeptides (e.g., single-chain polypeptides, antibodies, andantigen-binding fragments) described herein include those thatspecifically bind to TNFR2 with a K_(D) value of less than 100 nM (e.g.,95 nM, 90 nM, 85 nM, 80 nM, 75 nM, 70 nM, 65 nM, 60 nM, 55 nM, 50 nM, 45nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2nM, or 1 nM). In some embodiments, polypeptides (e.g., single-chainpolypeptides, antibodies, antigen-binding fragments, and constructsthereof) described herein specifically bind to TNFR2 with a K_(D) valueof less than 1 nM (e.g., (e.g., 990 pM, 980 pM, 970 pM, 960 pM, 950 pM,940 pM, 930 pM, 920 pM, 910 pM, 900 pM, 890 pM, 880 pM, 870 pM, 860 pM,850 pM, 840 pM, 830 pM, 820 pM, 810 pM, 800 pM, 790 pM, 780 pM, 770 pM,760 pM, 750 pM, 740 pM, 730 pM, 720 pM, 710 pM, 700 pM, 690 pM, 680 pM,670 pM, 660 pM, 650 pM, 640 pM, 630 pM, 620 pM, 610 pM, 600 pM, 590 pM,580 pM, 570 pM, 560 pM, 550 pM, 540 pM, 530 pM, 520 pM, 510 pM, 500 pM,490 pM, 480 pM, 470 pM, 460 pM, 450 pM, 440 pM, 430 pM, 420 pM, 410 pM,400 pM, 390 pM, 380 pM, 370 pM, 360 pM, 350 pM, 340 pM, 330 pM, 320 pM,310 pM, 300 pM, 290 pM, 280 pM, 270 pM, 260 pM, 250 pM, 240 pM, 230 pM,220 pM, 210 pM, 200 pM, 190 pM, 180 pM, 170 pM, 160 pM, 150 pM, 140 pM,130 pM, 120 pM, 110 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40pM, 30 pM, 20 pM, 10 pM, 5 pM, or 1 pM).

Polypeptides described herein can also be characterized by a variety ofin vitro binding assays. Examples of experiments that can be used todetermine the K_(D) or IC₅₀ of an anti-TNFR2 polypeptide include, e.g.,surface plasmon resonance, isothermal titration calorimetry,fluorescence anisotropy, and ELISA-based assays, among others. ELISArepresents a particularly useful method for analyzing antibody activity,as such assays typically require minimal concentrations of antibodies. Acommon signal that is analyzed in a typical ELISA assay is luminescence,which is typically the result of the activity of a peroxidase conjugatedto a secondary antibody that specifically binds a primary antibody(e.g., a TNFR2 antibody described herein). Polypeptides (e.g.,single-chain polypeptides, antibodies, and antigen-binding fragments)described herein are capable of binding TNFR2 and epitopes therein, suchas epitopes containing one or more continuous or discontinuous residueswithin CRD3 and/or CRD4 of human TNFR2. Antagonistic polypeptidesdescribed herein may additionally bind isolated peptides derived fromTNFR2 that structurally pre-organize various residues in a manner thatsimulates the conformation of the above epitopes in the native protein.For instance, polypeptides (e.g., single-chain polypeptides, antibodies,antigen-binding fragments, and constructs thereof) described herein maybind peptides containing the amino acid sequence of any one of SEQ IDNOs: 11, 19, 20, and 34-117, or a peptide having up to five amino acidsubstitutions with respect to the amino acid sequence of any one of SEQID NOs: 11, 19, 20, and 34-117 (such as a peptide having up to fiveconservative amino acid substitutions with respect to the amino acidsequence of any one of SEQ ID NOs: 11, 19, 20, and 34-117), and/or apeptide having an amino acid sequence that is at least 85% identical(e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to theamino acid sequence of any one of SEQ ID NOs: 11, 19, 20, and 34-117. Ina direct ELISA experiment, this binding can be quantified, e.g., byanalyzing the luminescence that occurs upon incubation of an HRPsubstrate (e.g., 2,2′-azino-di-3-ethylbenzthiazoline sulfonate) with anantigen-antibody complex bound to a HRP-conjugated secondary antibody.

Kinetic Properties of Antagonistic TNFR2 Polypeptides

In addition to the thermodynamic parameters of a TNFR2-polypeptideinteraction, it is also possible to quantitatively characterize thekinetic association and dissociation of a polypeptide described hereinwith TNFR2. This can be done, e.g., by monitoring the rate ofpolypeptide-antigen (e.g., antibody-antigen) complex formation accordingto established procedures. For example, one can use surface plasmonresonance (SPR) to determine the rate constants for the formation(k_(on)) and dissociation (k_(off)) of an antibody-TNFR2 complex. Thesedata also enable calculation of the equilibrium constant of (K_(D)) ofantibody-TNFR2 complex dissociation, since the equilibrium constant ofthis unimolecular dissociation can be expressed as the ratio of thek_(off) to k_(on) values. SPR is a technique that is particularlyadvantageous for determining kinetic and thermodynamic parameters ofreceptor-antibody interactions since the experiment does not requirethat one component be modified by attachment of a chemical label.Rather, the receptor is typically immobilized on a solid metallicsurface which is treated in pulses with solutions of increasingconcentrations of antibody. Antibody-receptor binding induces distortionin the angle of reflection of incident light at the metallic surface,and this change in refractive index over time as antibody is introducedto the system can be fit to established regression models in order tocalculate the association and dissociation rate constants of anantibody-receptor interaction.

Polypeptides (e.g., single-chain polypeptides, antibodies,antigen-binding fragments, and constructs thereof) described herein mayexhibit high k_(on) and low k_(off) values upon interaction with TNFR2,consistent with high-affinity receptor binding. For example,polypeptides described herein may exhibit k_(on) values in the presenceof TNFR2 of greater than 10⁴ M⁻¹ s⁻¹ (e.g., 1.0×10⁴ M⁻¹ s⁻¹, 1.5×10⁴ M⁻¹s⁻¹, 2.0×10⁴ M⁻¹ s⁻¹, 2.5×10⁴ M⁻¹ s⁻¹, 3.0×10⁴ M⁻¹ s⁻¹, 3.5×10⁴ M⁻¹ s⁻¹,4.0×10⁴ M⁻¹ s⁻¹, 4.5×10⁴ M⁻¹ s⁻¹, 5.0×10⁴ M⁻¹ s⁻¹, 5.5×10⁴ M⁻¹ s⁻¹,6.0×10⁴ M⁻¹ s⁻¹, 6.5×10⁴ M⁻¹ s⁻¹, 7.0×10⁵ M⁻¹ s⁻¹, 7.5×10⁵ M⁻¹ s⁻¹,8.0×10⁵ M⁻¹ s⁻¹, 8.5×10⁵ M⁻¹ s⁻¹, 9.0×10⁵ M⁻¹ s⁻¹, 9.5×10⁵ M⁻¹ s⁻¹,1.0×10⁵ M⁻¹ s⁻¹, 1.5×10⁵ M⁻¹ s⁻¹, 2.0×10⁵ M⁻¹ s⁻¹, 2.5×10⁵ M⁻¹ s⁻¹,3.0×10⁵ M⁻¹ s⁻¹, 3.5×10⁵ M⁻¹ s⁻¹, 4.0×10⁵ M⁻¹ s⁻¹, 4.5×10⁵ M⁻¹ s⁻¹,5.0×10⁵ M⁻¹ s⁻¹, 5.5×10⁵ M⁻¹ s⁻¹, 6.0×10⁵ M⁻¹ s⁻¹, 6.5×10⁵ M⁻¹ s⁻¹,7.0×10⁵ M⁻¹ s⁻¹, 7.5×10⁵ M⁻¹ s⁻¹, 8.0×10⁵ M⁻¹ s⁻¹, 8.5×10⁵ M⁻¹ s⁻¹,9.0×10⁵ M⁻¹ s⁻¹, 9.5×10⁵ M⁻¹ s⁻¹, or 1.0×10⁶ M⁻¹ s⁻¹). Polypeptides(e.g., single-chain polypeptides, antibodies, antigen-binding fragments,and constructs thereof) described herein may exhibit low k_(off) valueswhen bound to TNFR2, as these polypeptides are capable of interactingwith distinct TNFR2 epitopes with a high affinity. Residues within theseepitopes may form strong intermolecular contacts with TFNR2, which canslow the dissociation of the antibody-TNFR2 complex. This high receptoraffinity can manifest in low k_(off) values. For instance, polypeptidesdescribed herein may exhibit k_(off) values of less than 10⁻³ s⁻¹ whencomplexed to TNFR2 (e.g., 1.0×10⁻³ s⁻¹, 9.5×10⁻⁴ s⁻¹, 9.0×10⁻⁴ s⁻¹,8.5×10⁻⁴ s⁻¹, 8.0×10⁻⁴ s⁻¹, 7.5×10⁻⁴ s⁻¹, 7.0×10⁻⁴ s⁻¹, 6.5×10⁻⁴ s⁻¹,6.0×10⁻⁴ s⁻¹, 5.5×10⁻⁴ s⁻¹, 5.0×10⁻⁴ s⁻¹, 4.5×10⁻⁴ s⁻¹, 4.0×10⁻⁴ s⁻¹,3.5×10⁻⁴ s⁻¹, 3.0×10⁻⁴ s⁻¹, 2.5×10⁻⁴ s⁻¹, 2.0×10⁻⁴ s⁻¹, 1.5×10⁻⁴ s⁻¹,1.0×10⁻⁴ s⁻¹, 9.5×10⁻⁵ s⁻¹, 9.0×10⁻⁵ s⁻¹, 8.5×10⁻⁵ s⁻¹, 8.0×10⁻⁵ s⁻¹,7.5×10⁻⁵ s⁻¹, 7.0×10⁻⁵ s⁻¹, 6.5×10⁻⁵ s⁻¹, 6.0×10⁻⁵ s⁻¹, 5.5×10⁻⁵ s⁻¹,5.0×10⁻⁵ s⁻¹, 4.5×10⁻⁵ s⁻¹, 4.0×10⁻⁵ s⁻¹, 3.5×10⁻⁵ s⁻¹, 3.0×10⁻⁵ s⁻¹,2.5×10⁻⁵ s⁻¹, 2.0×10⁻⁵ s⁻¹, 1.5×10⁻⁵ s⁻¹, or 1.0×10⁻⁵ s⁻¹).

Epitopes within TNFR2 Bound by Antagonistic TNFR2 Polypeptides

Among the difficulties in developing anti-TNFR2 polypeptides (e.g.,single-chain polypeptides, antibodies, and antigen-binding fragments)that are capable of antagonizing TNFR2 has been the elucidation ofepitopes within TNFR2 that participate in antagonistic complex formationrather than epitopes that promote signal transduction. The presentdisclosure is based, in part, on the discovery of epitopes within TNFR2that, when bound, promote receptor antagonism and the ability to promoteone or more, or all, of the following advantageous biologicalactivities:

-   -   (a) Suppression of the proliferation of, and/or the direct        killing of, T-reg cells, for instance, by binding and        inactivating TNFR2 on the T-reg cell surface;    -   (b) Suppression of the proliferation of, and/or the direct        killing of, MDSCs, for instance, by binding and inactivating        TNFR2 on the MDSC surface;    -   (c) Promotion of the expansion of T effector cells, such as CD8+        T cells; and/or    -   (d) Suppression of the proliferation of, and/or the direct        killing of, TNFR2-expressing cancer cells, such as Hodgkin's        lymphoma cells, cutaneous non-Hodgkin's lymphoma cells, T cell        lymphoma cells, ovarian cancer cells, colon cancer cells,        multiple myeloma cells, renal cell carcinoma cells, skin cancer        cells, lung cancer cells, liver cancer cells, endometrial cancer        cells, hematopoietic or lymphoid cancer cells, central nervous        system cancer cells, breast cancer cells, pancreatic cancer        cells, stomach cancer cells, esophageal cancer cells, and upper        gastrointestinal cancer cells.

Antagonistic TNFR2 polypeptides, such dominant antagonistic TNFR2polypeptides (e.g., single-chain polypeptides, antibodies,antigen-binding fragments, and constructs thereof) described herein mayspecifically bind one or more of the following epitopes on human TNFR2:

-   -   (a) amino acids 142-146 of SEQ ID NO: 7 (KCRPG);    -   (b) amino acids 142-149 of SEQ ID NO: 7 (KCRPGFGV);    -   (c) amino acids 137-144 of SEQ ID NO: 7 (CAPLRKCR);    -   (d) amino acids 150-190 of SEQ ID NO: 7        (RPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAI);    -   (e) amino acids 161-169 of SEQ ID NO: 7 (CKPCAPGTF);    -   (f) amino acids 75-128 of SEQ ID NO: 7        (CDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCAL),        optionally in which the epitope is within amino acids 80-86        (DSTYTQL), 91-98 (PECLSCGS), or 116-123 (RICTCRPG) of SEQ ID NO:        7;    -   (g) amino acids 174-184 (SSTDICRPHQI) of SEQ ID NO: 7;    -   (h) amino acids 126-140 (CALSKQEGCRLCAPL) of SEQ ID NO: 7;    -   (i) amino acids 156-165 (TSDVVCKPCA) of SEQ ID NO: 7;    -   (j) an equivalent epitope within TNFR2 of a non-human mammal,        such as a non-human mammal described herein, an epitope that        exhibits at least 85% sequence identity (e.g., 85%, 90%, 95%,        97%, 99%, or 100% sequence identity) to any of the foregoing        epitopes, and/or an epitope that contains one or more        conservative amino acid substitutions relative to these        epitopes.

In some embodiments, antagonistic TNFR2 polypeptides, such as dominantantagonistic TNFR2 polypeptides (e.g., single-chain polypeptides,antibodies, antigen-binding fragments, and constructs thereof) describedherein do not bind or more, or all, residues of 142-146 of SEQ ID NO: 7within human TNFR2 (KCRPG, SEQ ID NO: 19). Additionally, antagonisticTNFR2 polypeptides described herein distinctly do not exhibit specificbinding to an epitope containing residues 56-60 of SEQ ID NO: 7 withinhuman TNFR2 (KCSPG, SEQ ID NO: 12). Polypeptides that exhibit theability to bind one or more of the above epitopes within human TNFR2 andan epitope containing residues 56-60 of SEQ ID NO: 7 within human TNFR2lack inhibitory (antagonistic) activity. As such, the ability of a TNFR2polypeptide to discriminate among these epitopes and specificallyinteract with one or more of the epitopes described above and to notengage in specific binding with an epitope composed of residues 56-60 ofSEQ ID NO: 7 within human TNFR2 characterizes polypeptides describedherein that antagonize TNFR2 signaling.

One exemplary procedure that can be used to predict the inhibitoryactivity of a TNFR2 polypeptide described herein is to determine theaffinity of the antibody or antibody fragment for a peptide containingthe KCRPG motif (SEQ ID NO: 19), such as a linear or cyclic peptide thatcontains this motif. The peptide may be, for example, structurallypre-organized by virtue of one or more conformational constraints (e.g.,backbone or side-chain-to-side-chain cyclization) in a manner thatsimulates the three-dimensional orientation of the KCRPG motif (SEQ IDNO: 19). For instance, antagonistic TNFR2 polypeptides described hereinmay specifically bind such a peptide with an affinity that is greaterthan that of the antagonistic TNFR2 polypeptide for a peptide fragmentdefined by residues 48-67 of SEQ ID NO: 7 within human TNFR2(QTAQMCCSKCSPGQHAKVFC, SEQ ID NO: 18). For example, antagonistic TNFR2polypeptides described herein may bind a peptide containing the KCRPGmotif (SEQ ID NO: 19) with an affinity that is, e.g., 10-fold, 15-fold,20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold,100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold,800-fold, 900-fold, 1000-fold, or more than 1000-fold greater than theaffinity of the antagonistic polypeptide for a peptide having the aminoacid sequence of SEQ ID NO: 18.

Antagonistic TNFR2 Polypeptides that Bind TNFR2 from Non-Human Animals

In addition to binding the epitopes detailed above within human TFNR2,antagonistic TNFR2 polypeptides described herein, such as dominantantagonistic TNFR2 polypeptides, also include those that specificallybind epitopes containing one or more equivalent motifs within TNFR2 froma non-human animal. The locations of epitopes equivalent those withinhuman TNFR2 that give rise to an antagonistic phenotype upon binding aredescribed, e.g., in WO 2016/187068 and WO 2017/197331, the disclosuresof which are incorporated herein by reference in their entirety.Exemplary TNFR2 proteins of non-human animals that may be bound byantagonistic polypeptides of the present disclosure include, withoutlimitation, TNFR2 proteins from cattle, bison, and other agriculturalanimals described herein.

The Antagonistic TNFR2 Antibody TNFRAB1

Exemplary antagonistic TNFR2 polypeptides described herein, such assingle-chain polypeptides, antibodies, antigen-binding fragmentsthereof, and constructs thereof, may include one or more, or all, of theCDRs of TNFRAB1, a murine antibody that antagonizes the TNFRα-TNFR2interaction. For instance, the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2,and/or CDR-L3 of TNFRAB1, and variants of these CDRs (e.g., variantsthat exhibit conservative amino acid substitutions relative to these CDRsequences) can be used to generate an antagonistic TNFR2 antibody orantigen-binding fragment thereof, for instance, using antibodyhumanization methods described herein or known in the art.

Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides,antibodies, and antigen-binding fragments) of the disclosure may exhibitbinding properties that are the same as or similar to those of TNFRAB1.These properties are as follows: In the presence of TNFR2, TNFRAB1exhibits a high k_(on) value of 4.98×10⁶ M⁻¹ s⁻¹, as well as a lowk_(off) of 2.21×10⁻⁴ s⁻¹ and a K_(D) of about 44.4 pM in complex withTNFR2. The KCRPGFGV motif (SEQ ID NO: 20), and specifically, the KCRPGsequence (SEQ ID NO: 19), has been identified as a particularlyimportant component of the functional epitope that establishesintermolecular contacts with TNFRAB1 as determined by epitope mappinganalysis. The interaction of these residues with anti-TNFR2 antibodiesof the disclosure selectively promotes antagonistic activity.Significantly, a TNFR2 epitope including amino acid residues 56-60 ofSEQ ID NO: 7 within human TNFR2 (KCSPG, SEQ ID NO: 12) is distinctly nota part of the conformational epitope that is specifically bound byTNFRAB1 or antagonistic TNFR2 antibodies or antibody fragments of thedisclosure, as specific binding to both of these epitopes has been shownto lead to a loss of, or significant reduction in, antagonisticactivity.

In addition to binding an epitope contained within the sequence KCRPGFGV(SEQ ID NO: 20), TNFRAB1 also binds to a downstream epitope containedwithin a sequence defined by positions 161-169 of SEQ ID NO: 7 withinhuman TNFR2 (CKPCAPGTF, SEQ ID NO: 21). TNFR2 antibodies and antibodyfragments of the disclosure may also bind this epitope or a largerregion within TNFR2 containing this epitope (e.g., a sequence thatincludes at least five continuous or discontinuous residues frompositions 150-190 of SEQ ID NO: 7 within human TNFR2(ARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAI, SEQ ID NO: 22). TNFRAB1contains two heavy chains, as well as two light chains. The heavy chainsof TNFRAB1 contain the following amino acid sequence (CDRs are indicatedin bold):

(SEQ ID NO: 2) EVQLQESGGGLVKPGGSLKLSCAASGFTFSSYVMSWVRQTPEKRLEWVATISSGGSYTYYPDSVKGRFTISRDNAKNTLYLQMSSLRSEDTAMYYCARQRVDGYSSYWYFDVWGAGTAVTVSS

The sequence of the TNFRAB1 light chain is as follows (CDRs areindicated in bold):

(SEQ ID NO: 4) DIVLTQSPAIMSASPGEKVTITCSASSSVYYMYWFQQKPGTSPKLWIYSTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQRRNYPYTFG GGTKLEIKRA

The heavy chain and light chain CDRs of TNFRAB2 are shown below:

TNFRAB1 CDR-H1: (SEQ ID NO: 23) GFTFSSY TNFRAB1 CDR-H2: (SEQ ID NO: 24)SSGGSY TNFRAB1 CDR-H3: (SEQ ID NO: 25) QRVDGYSSYWYFDV TNFRAB1 CDR-L1:(SEQ ID NO: 26) SASSSVYYMY TNFRAB1 CDR-L2: (SEQ ID NO: 26) STSNLASTNFRAB1 CDR-L3: (SEQ ID NO: 28) QQRRNYPYT

The Antagonistic TNFR2 Antibody TNFRAB2

Antagonistic TNFR2 polypeptides described herein, such as single-chainpolypeptides, antibodies, antigen-binding fragments thereof, andconstructs thereof, may include one or more, or all, of the CDRs ofTNFRAB2, another antibody that antagonizes the TNFRα-TNFR2 interaction.For instance, the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and/or CDR-L3of TNFRAB2, and variants of these CDRs (e.g., variants that exhibitconservative amino acid substitutions relative to these CDR sequences)can be used to generate an antagonistic TNFR2 antibody orantigen-binding fragment thereof, for instance, using antibodyhumanization methods described herein or known in the art.

For instance, antagonistic TNFR2 polypeptides (e.g., single-chainpolypeptides, antibodies, and antigen-binding fragments) of thedisclosure may exhibit binding properties that are the same as orsimilar to those of TNFRAB2. These properties are as follows: In thepresence of TNFR2, TNFRAB2 exhibits a high k_(on) value of 3.6099×10⁵M⁻¹ s⁻¹, as well as a low k_(off) of 2.24×10⁻⁴ s⁻¹ and a K_(D) of about621 pM in complex with TNFR2. An epitope containing residues 137-144 ofSEQ ID NO: 7 within human TNFR2 (CAPLRKCR, SEQ ID NO: 11) has beenidentified as a particularly important component of the functionalepitope that establishes intermolecular contacts with TNFRAB2 asdetermined by epitope mapping analysis. Included in the presentdisclosure are TNFR2 antibodies and antibody fragments that specificallybind this epitope.

In addition to binding an epitope containing residues CAPLRKCR (SEQ IDNO: 11), TNFRAB2 also binds to epitopes that include one or moreresidues within positions 80-86 of SEQ ID NO: 7 within human TNFR2(DSTYTQL, SEQ ID NO: 8), positions 91-98 of SEQ ID NO: 7 within humanTNFR2 (PECLSCGS, SEQ ID NO: 9), as well as positions 116-123 of SEQ IDNO: 7 within human TNFR2 (RICTCRPG, SEQ ID NO: 10). TNFR2 antibodies andantibody fragments of the disclosure may also bind one or more of theseepitopes. Antibodies and antibody fragments of the disclosure can bedesigned and identified using the knowledge of the epitopes specificallybound by TNFRAB2. For instance, one can use any of a variety of in vitropeptide display techniques or combinatorial antibody library screens asdescribed herein or known in the art in order to screen for antibodiescapable of binding these epitopes with high affinity and selectivity.

The heavy chain and light chain CDRs of TNFRAB2 are shown below:

TNFRAB2 CDR-H1: (SEQ ID NO: 274) GYTFTDYL TNFRAB2 CDR-H2:(SEQ ID NO: 258) VDPEYGST TNFRAB2 CDR-H3: (SEQ ID NO: 259)ARDDGSYSPFDYWG TNFRAB2 CDR-L1: (SEQ ID NO: 260) QNINKY TNFRAB2 CDR-L2:TYS TNFRAB2 CDR-L3: (SEQ ID NO: 272) CLQYVNLLT

Additionally, the CDR-L2 of TNFRAB2 is flanked by the N-terminalframework residues LLIR (SEQ ID NO: 262) and the C-terminal frameworkresidues TLE. Accordingly, antagonistic TNFR2 polypeptides (e.g.,single-chain polypeptides, antibodies, and antigen-binding fragments) ofthe disclosure include those that contain one or more of the above CDRsof TNFRAB2, as well as N-terminal LLIR (SEQ ID NO: 262) and C-terminalTLE residues that flank the CDR-L2 sequence of the antagonistic TNFR2antibody or antigen-binding fragment thereof.

The Antagonistic TNFR2 Antibody TNFRAB3

Antagonistic TNFR2 polypeptides described herein, such as single-chainpolypeptides, antibodies, antigen-binding fragments thereof, andconstructs thereof, may exhibit binding properties that are the same asor similar to those of TNFRAB3. TNFRAB3 is a monoclonal antibody and adominant TNFR2 antagonist. Monoclonal antibody TNFRAB3 has a CDR-H1amino acid sequence of GYTFTDVI (SEQ ID NO: 293). TNFRAB3 binds epitopeswithin CDR3 and/or CRD4 of human TNFR2 at the exclusion of epitopeswithin CRD1 of human TNFR2.

As described in detail below, antagonistic TNFR2 polypeptides describedherein (e.g., single-chain polypeptides, antibodies, antigen-bindingfragments thereof, and constructs thereof) can be generated by producingand identifying antibodies that exhibit epitope-binding propertiessimilar to those of TNFRAB3. Exemplary techniques for the production ofpolypeptides (e.g., single-chain polypeptides, antibodies,antigen-binding fragments thereof, and constructs thereof) that haveepitope-binding properties similar to those of TNFRAB3 include, withoutlimitation, the production of fully human, humanized, primatized, andchimeric antibodies that incorporate one or more, or all, of thecomplementarity-determining regions (CDRs) of TNFRAB3, as well asscreening for polypeptides that specifically bind one or more, or all,epitopes on TNFR2 that are specifically bound by TNFRAB3.

The Antagonistic TNFR2 Antibody TNFRAB4

Antagonistic TNFR2 polypeptides described herein, such as single-chainpolypeptides, antibodies, antigen-binding fragments thereof, andconstructs thereof, may exhibit binding properties that are the same asor similar to those of TNFRAB4. TNFRAB4 is a monoclonal murine antibodydescribed herein. This antibody is a dominant TNFR2 antagonist.Monoclonal antibody TNFRAB4 binds epitopes within human TNFR2 containingthe following amino acid residues:

-   -   (a) residues 174-184 of SEQ ID NO: 7 within human TNFR2        (SSTDICRPHQI, SEQ ID NO: 288);    -   (b) residues 126-140 of SEQ ID NO: 7 within human TNFR2        (CALSKQEGCRLCAPL), SEQ ID NO: 289); and    -   (c) residues 156-165 of SEQ ID NO: 7 within human TNFR2        (TSDVVCKPCA), SEQ ID NO: 290).

As described in detail below, antagonistic TNFR2 polypeptides describedherein (e.g., single-chain polypeptides, antibodies, antigen-bindingfragments thereof, and constructs thereof) can be generated by producingand identifying antibodies that exhibit epitope-binding propertiessimilar to those of TNFRAB4. Exemplary techniques for the production ofpolypeptides (e.g., single-chain polypeptides, antibodies,antigen-binding fragments thereof, and constructs thereof) that haveepitope-binding properties similar to those of TNFRAB4 include, withoutlimitation, the production of fully human, humanized, primatized, andchimeric antibodies that incorporate one or more, or all, of thecomplementarity-determining regions (CDRs) of TNFRAB4, as well asscreening for polypeptides that specifically bind one or more, or all,epitopes on TNFR2 that are specifically bound by TNFRAB4.

The Antagonistic TNFR2 Antibody TNFRAB5

Antagonistic TNFR2 polypeptides described herein, such as single-chainpolypeptides, antibodies, antigen-binding fragments thereof, andconstructs thereof, may exhibit binding properties that are the same asor similar to those of TNFRAB5. TNFRAB5 is a monoclonal antibody and adominant TNFR2 antagonist. Monoclonal antibody TNFRAB5 has a CDR-H1amino acid sequence of GYTFTDYS (SEQ ID NO: 294). TNFRAB5 binds epitopeswithin CDR3 and/or CRD4 of human TNFR2 at the exclusion of epitopeswithin CRD1 of human TNFR2.

As described in detail below, antagonistic TNFR2 polypeptides describedherein (e.g., single-chain polypeptides, antibodies, antigen-bindingfragments thereof, and constructs thereof) can be generated by producingand identifying antibodies that exhibit epitope-binding propertiessimilar to those of TNFRAB5. Exemplary techniques for the production ofpolypeptides (e.g., single-chain polypeptides, antibodies,antigen-binding fragments thereof, and constructs thereof) that haveepitope-binding properties similar to those of TNFRAB5 include, withoutlimitation, the production of fully human, humanized, primatized, andchimeric antibodies that incorporate one or more, or all, of thecomplementarity-determining regions (CDRs) of TNFRAB5, as well asscreening for polypeptides that specifically bind one or more, or all,epitopes on TNFR2 that are specifically bound by TNFRAB5.

Fully Human, Humanized, Primatized, and Chimeric Antibodies

Antibodies described herein include fully human, humanized, primatized,and chimeric antibodies that contain one or more, or all, of the CDRsequences of TNFRAB1, TNFRAB2, TNFRAB3, TNFRAB4, or TNFRAB5 (e.g., theCDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and/or CDR-L3 sequences ofTNFRAB1, TNFRAB2, TNFRAB3, TNFRAB4, or TNFRAB5). Additionally,antibodies described herein include fully human, humanized, primatized,and chimeric antibodies that contain one or more, or all, of the CDR-H1,CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences in which one ormore, or all, of the CDR sequences exhibits at least 85% sequenceidentity (e.g., 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity)to the corresponding CDR sequence of TNFRAB1, TNFRAB2, TNFRAB3, TNFRAB4,or TNFRAB5). Antagonistic TNFR2 antibodies described herein furtherinclude fully human, humanized, primatized, and chimeric antibodies thatcontain one or more, or all, of the CDR-H1, CDR-H2, CDR-H3, CDR-L1,CDR-L2, and CDR-L3 sequences in which one or more, or all, of the CDRsequences contains one or more (for instance, up to 3) amino acidsubstitutions (e.g., one or more conservative amino acid substitutions)relative to the corresponding CDR sequence of TNFRAB1, TNFRAB2, TNFRAB3,TNFRAB4, or TNFRAB5. For example, antagonistic TNFR2 antibodiesdescribed herein can be generated by incorporating any one or more ofthe CDR sequences of TNFRAB1, TNFRAB2, TNFRAB3, TNFRAB4, or TNFRAB5 intothe framework regions (e.g., FW1, FW2, FW3, and FW4) of a humanantibody. Exemplary framework regions that can be used for thedevelopment of a humanized anti-TNFR2 antibody containing one or more ofthe CDRs of TNFRAB1, TNFRAB2, TNFRAB3, TNFRAB4, or TNFRAB5 include,without limitation, those described in U.S. Pat. Nos. 7,732,578,8,093,068, and WO 2003/105782; the disclosures of each of which areincorporated herein by reference.

As an example, one strategy that can be used to design humanizedantibodies described herein is to align the sequences of the heavy chainvariable region and light chain variable region of TNFRAB1, TNFRAB2,TNFRAB3, TNFRAB4, or TNFRAB5 with the heavy chain variable region andlight chain variable region of a consensus human antibody. Consensushuman antibody heavy chain and light chain sequences are known in theart (see e.g., the “VBASE” human germline sequence database; see alsoKabat, et al., Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242, 1991; Tomlinson et al., J. Mol. Biol. 227:776-98, 1992; andCox et al, Eur. J. Immunol. 24:827-836, 1994; the disclosure of which isincorporated herein by reference). In this way, the variable domainframework residues and CDRs can be identified by sequence alignment (seeKabat, supra). One can substitute, for example, one or more of the CDRsof the consensus human antibody with the corresponding CDR(s) ofTNFRAB1, TNFRAB2, TNFRAB3, TNFRAB4, or TNFRAB5, in order to produce ahumanized TNFR2 antagonist antibody. Exemplary variable domains of aconsensus human antibody include the heavy chain variable domain:

(SEQ ID NO: 32) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAPGKGLEWVAVISENGSDTYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCAR DRGGAVSYFDVWGQGTLVTVSSand the light chain variable domain:

(SEQ ID NO: 33) DIQMTQSPSSLSASVGDRVTITCRASQDVSSYLAWYQQKPGKAPKLLIYAASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSLPYTF GQGTKVEIKRTidentified in U.S. Pat. No. 6,054,297; the disclosure of which isincorporated herein by reference (CDRs are shown in bold were determinedaccording to the method of Chothia, et al., J. Mol. Biol, 196:901-917,1987). These amino acid substitutions can be made, for example, byrecombinant expression of polynucleotides encoding the heavy and lightchains of a humanized antibody in a host cell using methods known in theart or described herein.

Similarly, this strategy can also be used to produce primatizedantagonistic TNFR2 antibodies, as one can substitute, for example, oneor more, or all, of the CDRs of a primate antibody consensus sequencewith, for example, one or more, or all, of the CDRs of TNFRAB1, TNFRAB2,TNFRAB3, TNFRAB4, or TNFRAB5. Consensus primate antibody sequences knownin the art (see e.g., U.S. Pat. Nos. 5,658,570; 5,681,722; and5,693,780; the disclosures of each of which are incorporated herein byreference).

In some embodiments, it may be desirable to import particular frameworkresidues in addition to CDR sequences from an antagonistic TNFR2antibody, such as TNFRAB1, TNFRAB2, TNFRAB3, TNFRAB4, or TNFRAB5, intothe heavy and/or light chain variable domains of a human antibody. Forinstance, U.S. Pat. No. 6,054,297 identifies several instances when itmay be advantageous to retain certain framework residues from aparticular antibody heavy chain or light chain variable region in theresulting humanized antibody. In some embodiments, framework residuesmay engage in non-covalent interactions with the antigen and thuscontribute to the affinity of the antibody for the target antigen. Insome embodiments, individual framework residues may modulate theconformation of a CDR, and thus indirectly influence the interaction ofthe antibody with the antigen. Certain framework residues may form theinterface between VH and VL domains, and may therefore contribute to theglobal antibody structure. In some cases, framework residues mayconstitute functional glycosylation sites (e.g., Asn-X-Ser/Thr) whichmay dictate antibody structure and antigen affinity upon attachment tocarbohydrate moieties. In cases such as those described above, it may bebeneficial to retain certain framework residues of a TNFR2 antagonistantibody (e.g., TNFRAB1, TNFRAB2, TNFRAB3, TNFRAB4, or TNFRAB5 in, e.g.,a humanized or primatized antagonistic antibody or antigen-bindingfragment thereof, as various framework residues may promote high epitopeaffinity and improved biochemical activity of the antibody orantigen-binding fragment thereof.

Antibodies described herein also include antibody fragments, Fabdomains, F(ab′) molecules, F(ab′)₂ molecules, single-chain variablefragments (scFvs), tandem scFv fragments, diabodies, triabodies, dualvariable domain immunoglobulins, multi-specific antibodies, bispecificantibodies, and heterospecific antibodies that contain one or more, orall, of the CDRs of TNFRAB1, TNFRAB2, TNFRAB3, TNFRAB4, or TNFRAB5, orone or more, or all, of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, andCDR-L3 sequences in which one or more, or all, of the CDR sequencesexhibits at least 85% sequence identity (e.g., 90%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity) to the corresponding CDR sequence ofTNFRAB1, TNFRAB2, TNFRAB3, TNFRAB4, or TNFRAB5). Antagonistic TNFR2antibodies described herein further include fully human, humanized,primatized, and chimeric antibodies that contain one or more, or all, ofthe CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences inwhich one or more, or all, of the CDR sequences contains one or more(for instance, up to 3) amino acid substitutions (e.g., one or moreconservative amino acid substitutions) relative to the corresponding CDRsequence of TNFRAB1, TNFRAB2, TNFRAB3, TNFRAB4, or TNFRAB5. Thesemolecules can be expressed recombinantly, e.g., by incorporatingpolynucleotides encoding these proteins into expression vectors fortransfection in a eukaryotic or prokaryotic cell using techniquesdescribed herein or known in the art, or synthesized chemically, e.g.,by solid phase peptide synthesis methods described herein or known inthe art.

Polypeptides described herein additionally include antibody-likescaffolds that contain, for example, one or more, or all, of the CDRs ofTNFRAB1, TNFRAB2, TNFRAB3, TNFRAB4, or TNFRAB5, or one or more, or all,of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences inwhich one or more, or all, of the CDR sequences exhibits at least 85%sequence identity (e.g., 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity) to the corresponding CDR sequence of TNFRAB1, TNFRAB2,TNFRAB3, TNFRAB4, or TNFRAB5) or contains one or more (for instance, upto 3) amino acid substitutions (e.g., one or more conservative aminoacid substitutions) relative to the corresponding CDR sequence ofTNFRAB1, TNFRAB2, TNFRAB3, TNFRAB4, or TNFRAB5. Examples ofantibody-like scaffolds include proteins that contain a tenthfibronectin type III domain (¹⁰Fn3), which contains BC, DE, and FGstructural loops analogous to canonical antibodies. The tertiarystructure of the ¹⁰Fn3 domain resembles that of the variable region ofthe IgG heavy chain, and one of skill in the art can graft, e.g., one ormore, or all, of the CDR sequences of TNFRAB1, TNFRAB2, TNFRAB3,TNFRAB4, or TNFRAB5 or sequences having at least 85% sequence identity(e.g., 90%, 95%, 97%, 99%, or 100% sequence identity) to any one or moreof these CDR sequences or sequences containing amino acid substitutions,such as conservative or nonconservative amino acid substitutions (e.g.,up to 3 amino acid substitutions) relative to one or more of these CDRsequences onto the fibronectin scaffold by replacing residues of the BC,DE, and FG loops of ¹⁰Fn3 with residues of the corresponding CDRsequence of TNFRAB1, TNFRAB2, TNFRAB3, TNFRAB4, or TNFRAB5. This can beachieved by recombinant expression of a modified ¹⁰Fn3 domain in aprokaryotic or eukaryotic cell (e.g., using the vectors and techniquesdescribed herein). Examples of using the ¹⁰Fn3 domain as anantibody-like scaffold for the grafting of CDRs from antibodies onto theBC, DE, and FG structural loops are reported in WO 2000/034784, WO2009/142773, WO 2012/088006, and U.S. Pat. No. 8,278,419; thedisclosures of each of which are incorporated herein by reference.

Molecular Determinants of TNFR2 Affinity and Antagonism

The polypeptides of the disclosure may exhibit a series of sharedstructural features that give rise to a TNFR2 antagonist phenotype(e.g., a dominant TNFR2 antagonist phenotype). For example, alignment ofthe amino acid sequences of the CDR-H1 of each of TNRAB1, TNFRAB2,TNFRAB3, and TNFRAB5 demonstrate that these antibodies feature aconserved consensus sequence, as shown below:

(TNFRAB1 CDR-H1, SEQ ID NO: 23) G F T F S S Y(TNFRAB2 CDR-H1, SEQ ID NO: 274) G Y T F T D Y L(TNFRAB3 CDR-H1, SEQ ID NO: 293) G Y T F T D V I(TNFRAB5 CDR-H1, SEQ ID NO: 294) G Y T F T D Y S(Consensus sequence, SEQ ID NO: 295) G (Y/F) T F (S/T) — Y —

Alignment of the sequences reveals a shared GXTFXXYX motif, in which “X”independently designates any amino acid, such as an aromatic residue(e.g., a tyrosine or phenylalanine residue) at position two, a polar,uncharged residue (e.g., a serine or threonine residue) at positionfive, a polar, optionally negatively charged residue (e.g., a serine,aspartic acid, or glutamic acid residue) at position six, and a leucine,isoleucine, or serine residue at position seven. It has been discoveredthat, despite originating from a variety of different animal types byway of immunization, surprisingly, antagonistic TNFR2 polypeptides thatexhibit the phenotypes described herein exhibit a shared CDR-H1 coresequence.

Additionally, sequence analysis of the CDR-H2 regions of TNFRAB1 andTNFRAB2 similarly reveals a set of conserved amino acids at variouspositions throughout these regions:

(TNFRAB1 CDR-H2, SEQ ID NO: 24) SSG--GSY(TNFRAB2 CDR-H2, SEQ ID NO: 258) VDPEYGST (Consensus sequence) -----GS-

Analysis of this sequence alignment demonstrates that the CDR-H2sequences exhibit a conserved GS motif at the C-terminal end of theCDR-H2 region, with side-chains of variable molecular size, polarity,and electrostatic charge tolerated at the remaining positions.

The CDR-H3 regions of antagonistic TNFR2 antibodies show a similarconvergence towards a consensus amino acid sequence. Shown below are theCDR-H3 sequences of TNFRAB1, TNFRAB2, and TNFR2A3, another monoclonalantagonistic TNFR2 antibody. TNFR2A3 is described in WO 2017/197331, thedisclosure of which is incorporated herein by reference in its entirety.An analysis of the residues common to the CDR-H3 sequences of theseantibodies provides insight into the molecular features of antibodiesthat bind TNFR2 and exhibit an antagonistic effect, such as a dominantantagonistic effect. Epitope mapping analysis has shown that bothTNFRAB1, TNFRAB2, and TNFR2A3 bind epitopes within TNFR2 that containresidues 142-146 of SEQ ID NO: 7 and do not bind epitopes containingresidues 56-60 of SEQ ID NO: 7. The structural similarities betweencorresponding CDR-H3 regions provide a basis for predicting residuesubstitutions that may preserve or enhance TNFR2 affinity and antagonism(e.g., dominant antagonism). The CDR-H3 sequences of TNFRAB1, TNFRAB2,and TNFR2A3 are shown below:

(TNFRAB1 CDR-H3, SEQ ID NO: 25) QRVDGYSSYWYFDV(TNFRAB2 CDR-H3, SEQ ID NO: 259) ARDDG-S-YSPFDYWG(TNFR2A3 CDR-H3, SEQ ID NO: 284) ARDDG-S-YSPFDYFG (Consensus sequence)-R-DG-S-Y--FD---

Inspection of the CDR-H3 sequences of TNFRAB1, TNFRAB2, and TNFR2A3reveals conserved arginine, aspartic acid, glycine, serine, tyrosine,and phenylalanine residues throughout this CDR. Notably, residues ofvarying steric and electrostatic properties are tolerated in theremaining positions. For instance, the first position of the CDR-H3sequence tolerates amino acid residues of contrasting size and hydrogenbond-forming tendencies, as the first position of CDR-H3 in TNFRAB1features a polar glutamine residue containing a carboxamide side-chainwith hydrogen bond donor and acceptor moieties, while an alanine residuebearing an unfunctionalized methyl side-chain is found at thecorresponding position in TNFRAB2 and TNFR2A3. Additionally, the thirdposition in the above CDR-H3 sequences features a hydrophobic valine inTNFRAB1 and an anionic aspartic acid moiety in the correspondingposition of TNFRAB2. Similarly, positions ten and eleven of the CDR-H3of TNFRAB1 contain aromatic systems, while the corresponding residues inTNFRAB2 and TNFR2A3 contain polar and cyclic aliphatic substituents.

A similar analysis reveals molecular features common to the CDR-Lsequences of TNFRAB1 and TNFRAB2. For instance, the CDR-L1 sequences ofTNFRAB1 and TNFRAB2 are shown below:

(TNFRAB1 CDR-L1, SEQ ID NO: 26) SASSSVYYMY(TNFRAB2 CDR-L1, SEQ ID NO: 260) Q-N--INK-Y (Consensus residue) Y

Inspection of these sequences reveals that a hydroxyl-containingtyrosine residue is featured at the final position of CDR-L1, whileresidues of varying physicochemical properties are tolerated at theremaining positions. Similarly, analysis of the CDR-L2 regions ofTNFRAB1 and TNFRAB2 reveals a conserved amino acid at the final positionin both regions:

(TNFRAB1 CDR-L2, SEQ ID NO: 27) STSNLAS (TNFRAB2 CDR-L2) YT----S(Consensus sequence) T----S

Analysis of the above sequence alignment demonstrates that serineresidues are featured at the third position of these CDR-L2 sequences,while substitutions are widely tolerated at the remaining residues.Similarly, the CDR-L3 sequences of TNRAB1 and TNFRAB2 are as follows:

(TNFRAB1 CDR-L3, SEQ ID NO: 28) Q-QRRNYPY------T(TNFRAB2 CDR-L3, SEQ ID NO: 261) CLQ---YVNL(L/I)T (Consensus sequence)--Q---Y--------T

Analysis of the CDR-L3 sequences of TNFRAB1 and TNFRAB2 reveals apreference for tyrosine and threonine residues at distinct positionswithin these regions, while amino acids of a wide range ofphysicochemical characteristics are tolerated at other positions,including residues with cationic side-chains (Arg), conformationallyrestricted side-chains (Pro), and side-chains of varying polarity (e.g.,Gln, Asn, Leu, and Val). Collectively, the shared structural features ofthe above CDR-H and CDR-L sequences provide insight into those residuesthat are important for selectively binding one or more residues of theKCRPG epitope of TNFR2 (positions 142-146 of SEQ ID NO: 7, shown in SEQID NO: 19) in an anti-parallel dimer configuration and demonstrate thatcertain amino acids can be varied while retaining affinity and dominantantagonistic activity.

Antagonistic TNFR2 polypeptides of the disclosure, such as dominantantagonistic TNFR2 polypeptides (e.g., single-chain polypeptides,antibodies, or antigen-binding fragments thereof) may therefore haveheavy chain and light chain CDRs that contain the above consensussequences. For instance, TNFR2 antagonists of the disclosure may have aCDR-H1 having the amino acid sequence Z⁴JZ³Z⁵(J)₂Z⁵J; a CDR-H2 havingthe amino acid sequence (J)₅Z⁴Z³J; a CDR-L1 having the amino acidsequence (J)₅Z⁵; a CDR-L2 having the amino acid sequence (J)₂Z³; and/ora CDR-L3 having the amino acid sequence (J)₃Z⁵(J)₄Z³; in which each J isindependently a naturally occurring amino acid; each Z¹ is independentlya naturally occurring amino acid containing a cationic side-chain atphysiological pH; each Z² is independently a naturally occurring aminoacid containing an anionic side-chain at physiological pH; each Z³ isindependently a naturally occurring amino acid containing a polar,uncharged side-chain at physiological pH; each Z⁴ is independently aglycine or alanine; and each Z⁵ is independently a naturally occurringamino acid containing a hydrophobic side-chain.

In some embodiments, antagonistic TNFR2 polypeptides of the disclosure,such as dominant antagonistic TNFR2 polypeptides (e.g., single-chainpolypeptides, antibodies, or antigen-binding fragments thereof) may havea CDR-H1 having the amino acid sequence GJTF(J)₂YJ (SEQ ID NO: 277); aCDR-H2 having the amino acid sequence (J)₅GSJ; a CDR-L1 having the aminoacid sequence (J)₅Y; a CDR-L2 having the amino acid sequence (J)₂S;and/or a CDR-L3 having the amino acid sequence (J)₃Y(J)₄T; in which eachJ is independently a naturally occurring amino acid.

Antagonistic TNFR2 polypeptides of the disclosure, such as dominantantagonistic TNFR2 polypeptides (e.g., single-chain polypeptides,antibodies, or antigen-binding fragments thereof) may have a CDR-H1having the amino acid sequence Z⁴YZ³Z⁵TDZ⁵X; a CDR-H2 having the aminoacid sequence VDPEYZ⁴Z³T (SEQ ID NO: 264); a CDR-L1 having the aminoacid sequence QNINKZ⁵ (SEQ ID NO: 268); a CDR-L2 having the amino acidsequence TYZ³ or YTZ³; and/or a CDR-L3 having the amino acid sequenceCLQZ⁵VNLXZ³ (SEQ ID NO: 271); in which each Z¹ is independently an aminoacid containing a cationic side-chain at physiological pH; each Z² isindependently an amino acid containing an anionic side-chain atphysiological pH; each Z³ is independently an amino acid containing apolar, uncharged side-chain at physiological pH; each Z⁴ isindependently a glycine or alanine; each Z⁵ is independently an aminoacid containing a hydrophobic side-chain; and each X is independentlyleucine or isoleucine.

In some embodiments, antagonistic TNFR2 polypeptides of the disclosure,such as dominant antagonistic TNFR2 polypeptides (e.g., single-chainpolypeptides, antibodies, or antigen-binding fragments thereof) may havea CDR-H1 having the amino acid sequence GYTFTDYX (SEQ ID NO: 257), or anamino acid sequence having up to two amino acid substitutions relativeto this sequence; a CDR-H2 having the amino acid sequence VDPEYGST (SEQID NO: 258), or an amino acid sequence having up to two amino acidsubstitutions relative to this sequence; a CDR-L1 having the amino acidsequence QNINKY (SEQ ID NO: 260), or an amino acid sequence having up totwo amino acid substitutions relative to this sequence; a CDR-L2 havingthe amino acid sequence TYS or YTS; and/or a CDR-L3 having the aminoacid sequence CLQYVNLXT (SEQ ID NO: 261), or an amino acid sequencehaving up to two amino acid substitutions relative to this sequence.

For example, in some embodiments, antagonistic TNFR2 polypeptides of thedisclosure, such as dominant antagonistic TNFR2 polypeptides (e.g.,single-chain polypeptides, antibodies, or antigen-binding fragmentsthereof) may have a CDR-H1 having the amino acid sequence GYTFTDYL (SEQID NO: 274), or an amino acid sequence having up to two amino acidsubstitutions relative to this sequence; and a CDR-L3 having the aminoacid sequence CLQYVNLIT (SEQ ID NO: 273), or an amino acid sequencehaving up to two amino acid substitutions relative to this sequence. Thepresent disclosure is based in part on the discovery that thisparticular combination of CDR-H1 and CDR-L3 regions promote theselective killing of activated T-reg cells and potentiate augmented Teffector cell proliferation. As described herein, these phenotypes arebeneficial for the treatment of cancers and infectious diseases, as theability to deplete activated T-reg cell populations in a patientsuffering from such pathologies can lessen the attenuation of cytotoxicCD8+ T cells, thereby enabling effector cells to mount an immuneresponse against cancerous and infectious cells.

Exemplary Humanized TNFR2 Antibodies and Antigen-Binding FragmentsThereof

Examples of humanized antagonistic TNFR2 antibodies and antigen-bindingfragments thereof of the disclosure include those having a heavy chainand/or light chain shown in Table 1, which is reproduced below. Asdescribed in further detail in the Examples, the antagonistic TNFR2antibodies described in Table 1, above, were developed by humanizationof murine monoclonal antibodies using humanization techniques describedherein. In addition to humanization, the antibodies shown in Table 1incorporate C232S and C233S substitutions within the immunoglobulinhinge region. As described above, these substitutions confer a varietyof beneficial properties to antagonistic TNFR2 antibodies, including anelevated inhibitory effect on TNFR2 signalling (and, thus, heightenedTreg depletion and effector T cell proliferation). Exemplaryantagonistic TNFR2 antibodies and antigen-binding fragments of thedisclosure include those shown in Table 1, above, as well as those thatcontain a heavy chain and/or light chain having at least 85% sequenceidentity (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to a heavychain and/or light chain shown in Table 1.

Examples of humanized antagonistic TNFR2 antibodies and antigen-bindingfragments thereof also include antibodies and antigen-binding fragmentscontaining one or more, or all, of the CDRs shown in Table 1, above, oranother CDR described herein. For example, humanized TNFR2 antibodies orantigen-binding fragments of the disclosure may contain a CDR-H1 havingthe amino acid sequence GJTF(J)₂Y (SEQ ID NO: 276) or GJTF(J)₂YJ (SEQ IDNO: 277), in which each J is independently a naturally occurring aminoacid. In some embodiments, the polypeptide (e.g., a single-chainpolypeptide, antibody, antigen-binding fragment thereof, or constructthereof) further contains:

-   -   (a) a CDR-H2 having the amino acid sequence (J)₃GSJ or (J)₅GSJ;    -   (b) a CDR-H3 having the amino acid sequence JRJDGJSJY(J)₂FDJ        (SEQ ID NO: 278) or JRJDGSY(J)₂FD(J)₃ (SEQ ID NO: 279);    -   (c) a CDR-L1 having the amino acid sequence (J)₉Y or (J)₅Y;    -   (d) a CDR-L2 having the amino acid sequence (J)₆S or (J)₂S;        and/or    -   (e) a CDR-L3 having the amino acid sequence (J)₅Y(J)₂T or        (J)₃Y(J)₄T,    -   in which each J is independently a naturally occurring amino        acid.

The humanized antibody or antigen-binding fragment thereof may contain aCDR-H1 having the amino acid sequence Z⁴FZ³Z⁵SSZ⁵ or Z⁴YZ³Z⁵TDZ⁵X;

-   -   In which each Z³ is independently an amino acid including a        polar, uncharged side-chain at physiological pH;    -   each Z⁴ is independently a glycine or alanine;    -   each Z⁵ is independently an amino acid including a hydrophobic        side-chain; and    -   each X is independently leucine or isoleucine.

In some embodiments, the humanized antibody or antigen-binding fragmentthereof further contains:

-   -   (a) a CDR-H2 having the amino acid sequence SSGZ⁴Z³Y (SEQ ID        NO: 263) or VDPEYZ⁴Z³T (SEQ ID NO: 264);    -   (b) a CDR-H3 having the amino acid sequence        QZ¹VZ²Z⁴YZ³SZ⁵WYZ⁵Z²Z⁵ (SEQ ID NO: 265) or        AZ¹DZ²Z⁴Z³Z⁵SPZ⁵Z²Z⁵WG (SEQ ID NO: 266);    -   (c) a CDR-L1 having the amino acid sequence SASSSVYYMZ⁵ (SEQ ID        NO: 267) or QNINKZ⁵ (SEQ ID NO: 268);    -   (d) a CDR-L2 having the amino acid sequence STSNLAZ³ (SEQ ID NO:        269), TYZ³, or YTZ³; and/or    -   (e) a CDR-L3 having the amino acid sequence QQRRNZ⁵PYZ³ (SEQ ID        NO: 270) or CLQZ⁵VNLXZ³ (SEQ ID NO: 271);    -   in which each Z¹ is independently an amino acid including a        cationic side-chain at physiological pH;    -   each Z² is independently an amino acid including an anionic        side-chain at physiological pH;    -   each Z³ is independently an amino acid including a polar,        uncharged side-chain at physiological pH;    -   each Z⁴ is independently a glycine or alanine;    -   each Z⁵ is independently an amino acid including a hydrophobic        side-chain; and    -   each X is independently leucine or isoleucine.

The humanized antibody or antigen-binding fragment thereof may contain aCDR-H1 having the amino acid sequence GFTFSSY (SEQ ID NO: 23), GYTFTDYX(SEQ ID NO: 257), or an amino acid sequence having up to two amino acidsubstitutions (e.g., conservative amino acid substitutions) relative tothese sequences, in which each X is independently leucine or isoleucine,optionally in which the amino acid substitutions are conservative aminoacid substitutions. In some embodiments, the humanized antibody orantigen-binding fragment thereof further contains:

-   -   (a) a CDR-H2 having the amino acid sequence SSGGSY (SEQ ID NO:        24), VDPEYGST (SEQ ID NO: 258), or an amino acid sequence having        up to two amino acid substitutions (e.g., conservative amino        acid substitutions) relative to these sequences;    -   (b) a CDR-H3 having the amino acid sequence QRVDGYSSYWYFDV (SEQ        ID NO: 25), ARDDGSYSPFDYWG (SEQ ID NO: 259), ARDDGSYSPFDY (SEQ        ID NO: 296), or an amino acid sequence having up to two amino        acid substitutions (e.g., conservative amino acid substitutions)        relative to these sequences;    -   (c) a CDR-L1 having the amino acid sequence SASSSVYYMY (SEQ ID        NO: 26), QNINKY (SEQ ID NO: 260), or an amino acid sequence        having up to two amino acid substitutions (e.g., conservative        amino acid substitutions) relative to these sequences;    -   (d) a CDR-L2 having the amino acid sequence STSNLAS (SEQ ID NO:        27), TYS, YTS, or an amino acid sequence having up to two amino        acid substitutions (e.g., conservative amino acid substitutions)        relative to SEQ ID NO: 27; and/or    -   (e) a CDR-L3 having the amino acid sequence QQRRNYPYT (SEQ ID        NO: 28), CLQYVNLXT (SEQ ID NO: 261), or an amino acid sequence        having up to two amino acid substitutions (e.g., conservative        amino acid substitutions) relative to these sequences.

In some embodiments, the humanized antibody or antigen-binding fragmentthereof contains a heavy chain including one or more of the followingCDRs:

-   -   (a) a CDR-H1 having the amino acid sequence GFTFSSY (SEQ ID NO:        23);    -   (b) a CDR-H2 having the amino acid sequence SSGGSY (SEQ ID NO:        24); and    -   (c) a CDR-H3 having the amino acid sequence QRVDGYSSYWYFDV (SEQ        ID NO: 25).

The humanized antibody or antigen-binding fragment thereof may contain,for example, a heavy chain having one or more of the following CDRs:

-   -   (a) a CDR-H1 having the amino acid sequence GYTFTDYX (SEQ ID NO:        257);    -   (b) a CDR-H2 having the amino acid sequence VDPEYGST (SEQ ID NO:        258); and    -   (c) a CDR-H3 having the amino acid sequence ARDDGSYSPFDYWG (SEQ        ID NO: 259);    -   in which each X is independently leucine or isoleucine.

In some embodiments, the CDR-H1 has the amino acid sequence GYTFTDYL(SEQ ID NO: 274). In some embodiments, the CDR-H1 has the amino acidsequence GYTFTDYI (SEQ ID NO: 275). In some embodiments, the CDR-H1 hasthe amino acid sequence GYTFTDVI (SEQ ID NO: 293). In some embodiments,the CDR-H1 has the amino acid sequence GYTFTDYS (SEQ ID NO: 294).

Additionally or alternatively, the humanized antibody or antigen-bindingfragment thereof may contain, for example, a light chain having one ormore of the following CDRs:

-   -   (a) a CDR-L1 having the amino acid sequence SASSSVYYMY (SEQ ID        NO: 26);    -   (b) a CDR-L2 having the amino acid sequence STSNLAS (SEQ ID NO:        27); and    -   (c) a CDR-L3 having the amino acid sequence QQRRNYPYT (SEQ ID        NO: 28).

In some embodiments, the humanized antibody or antigen-binding fragmentthereof contains a light chain having one or more of the following CDRs:

-   -   (a) a CDR-L1 having the amino acid sequence QNINKY (SEQ ID NO:        260);    -   (b) a CDR-L2 having the amino acid sequence TYS or YTS; and    -   (c) a CDR-L3 having the amino acid sequence CLQYVNLXT (SEQ ID        NO: 261);    -   in which each X is independently leucine or isoleucine.

In some embodiments, the CDR-L2 has the amino acid sequence TYS. In someembodiments, the CDR-L2 has the amino acid sequence YTS. The CDR-L3 mayhave the amino acid sequence CLQYVNLLT (SEQ ID NO: 272). In someembodiments, the CDR-L3 has the amino acid sequence CLQYVNLIT (SEQ IDNO: 273).

The humanized antibody or antigen-binding fragment thereof may containthree heavy chain CDRs, including:

-   -   (a) a CDR-H1 having the amino acid sequence GFTFSSY (SEQ ID NO:        23);    -   (b) a CDR-H2 having the amino acid sequence SSGGSY (SEQ ID NO:        24); and    -   (c) a CDR-H3 having the amino acid sequence QRVDGYSSYWYFDV (SEQ        ID NO: 25);    -   and may further contain three light chain CDRs, including:    -   (a) a CDR-L1 having the amino acid sequence SASSSVYYMY (SEQ ID        NO: 26);    -   (b) a CDR-L2 having the amino acid sequence STSNLAS (SEQ ID NO:        27); and    -   (c) a CDR-L3 having the amino acid sequence QQRRNYPYT (SEQ ID        NO: 28).

In some embodiments, the humanized antibody or antigen-binding fragmentthereof contains three heavy chain CDRs, including:

-   -   (a) a CDR-H1 having the amino acid sequence GYTFTDYX (SEQ ID NO:        257), such as GYTFTDYL (SEQ ID NO: 274) or GYTFTDYI (SEQ ID NO:        275), preferably GYTFTDYL (SEQ ID NO: 274);    -   (b) a CDR-H2 having the amino acid sequence VDPEYGST (SEQ ID NO:        258); and    -   (c) a CDR-H3 having the amino acid sequence ARDDGSYSPFDYWG (SEQ        ID NO: 259);    -   and further contains three light chain CDRs, including:    -   (d) a CDR-L1 having the amino acid sequence QNINKY (SEQ ID NO:        260);    -   (e) a CDR-L2 having the amino acid sequence TYS or YTS; and    -   (f) a CDR-L3 having the amino acid sequence CLQYVNLXT (SEQ ID        NO: 261), such as CLQYVNLLT (SEQ ID NO: 272) or CLQYVNLIT (SEQ        ID NO: 273), preferably CLQYVNLIT (SEQ ID NO: 273),    -   in which each X is independently leucine or isoleucine.    -   In some embodiments, the humanized antibody or antigen-binding        fragment thereof includes a framework region having the amino        acid sequence LLIR (SEQ ID NO: 262) bound to the N-terminus of        the CDR-L2 and/or a framework region having the amino acid        sequence TLE bound to the C-terminus of the CDR-L2.

Nucleic Acids and Expression Systems

Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides,antibodies, antigen-binding fragments thereof, and constructs thereof)described herein (e.g., any one or more of antibodies 1-25 described inTable 1 and variants thereof, such as an antibody or antigen-bindingfragment containing one or more, or all, of the CDRs set forth inTable 1) can be prepared by any of a variety of established techniques.For instance, an antagonistic TNFR2 antibody or antigen-binding fragmentthereof described herein can be prepared by recombinant expression ofimmunoglobulin light and heavy chain genes in a host cell. To express anantibody recombinantly, a host cell can be transfected with one or morerecombinant expression vectors carrying DNA fragments encoding theimmunoglobulin light and heavy chains of the antibody such that thelight and heavy chains are expressed in the host cell and, optionally,secreted into the medium in which the host cells are cultured, fromwhich medium the antibodies can be recovered. Standard recombinant DNAmethodologies are used to obtain antibody heavy and light chain genes,incorporate these genes into recombinant expression vectors andintroduce the vectors into host cells, such as those described inMolecular Cloning; A Laboratory Manual, Second Edition (Sambrook,Fritsch and Maniatis (eds), Cold Spring Harbor, N. Y., 1989), CurrentProtocols in Molecular Biology (Ausubel et al., eds., Greene PublishingAssociates, 1989), and in U.S. Pat. No. 4,816,397; the disclosures ofeach of which are incorporated herein by reference.

Vectors for Expression of Antagonistic TNFR2 Polypeptides

Viral genomes provide a rich source of vectors that can be used for theefficient delivery of exogenous genes into the genome of a cell (e.g., aeukaryotic or prokaryotic cell) and may be used to express a TNFR2antagonist polypeptide described herein (e.g., any one or more ofantibodies 1-25 described in Table 1 and variants thereof, such as anantibody or antigen-binding fragment containing one or more, or all, ofthe CDRs set forth in Table 1). Viral genomes are particularly usefulvectors for gene delivery because the polynucleotides contained withinsuch genomes are typically incorporated into the genome of a target cellby generalized or specialized transduction. These processes occur aspart of the natural viral replication cycle, and do not require addedproteins or reagents in order to induce gene integration. Examples ofviral vectors include a retrovirus, adenovirus (e.g., Ad5, Ad26, Ad34,Ad35, and Ad48), parvovirus (e.g., adeno-associated viruses),coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g.,influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitisvirus), paramyxovirus (e.g. measles and Sendai), positive strand RNAviruses, such as picornavirus and alphavirus, and double stranded DNAviruses including adenovirus, herpesvirus (e.g., Herpes Simplex virustypes 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g.,vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox). Otherviruses useful for delivering polynucleotides encoding antibody lightand heavy chains or antibody fragments described herein include Norwalkvirus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, andhepatitis virus, for example. Examples of retroviruses include: avianleukosis-sarcoma, mammalian C-type, B-type viruses, D-type viruses,HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: Theviruses and their replication, In Fundamental Virology, Third Edition,B. N. Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia,1996). Other examples include murine leukemia viruses, murine sarcomaviruses, mouse mammary tumor virus, bovine leukemia virus, felineleukemia virus, feline sarcoma virus, avian leukemia virus, human T cellleukemia virus, baboon endogenous virus, Gibbon ape leukemia virus,Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcomavirus, Rous sarcoma virus and lentiviruses. Other examples of vectorsare described, for example, in McVey et al., (U.S. Pat. No. 5,801,030);the disclosures of each of which are incorporated herein by reference.

Genome Editing Techniques

In addition to viral vectors, a variety of additional methods have beendeveloped for the incorporation of genes, e.g., those encoding antibodylight and heavy chains, single-chain polypeptides, single-chain variablefragments (scFvs), tandem scFvs, Fab domains, F(ab′)₂ domains,diabodies, and triabodies, among others, into the genomes of targetcells for polypeptide expression. One such method that can be used forincorporating polynucleotides encoding anti-TNFR2 polypeptides (e.g.,single-chain polypeptides, antibodies, antigen-binding fragmentsthereof, or constructs thereof) described herein (e.g., any one or moreof antibodies 1-25 described in Table 1 and variants thereof, such as anantibody or antigen-binding fragment containing one or more, or all, ofthe CDRs set forth in Table 1) into prokaryotic or eukaryotic cellsincludes transposons. Transposons are polynucleotides that encodetransposase enzymes and contain a polynucleotide sequence or gene ofinterest flanked by excision sites at the 5′ and 3′ positions. Once atransposon has been delivered into a cell, expression of the transposasegene commences and results in active enzymes that cleave the gene ofinterest from the transposon. This activity is mediated by thesite-specific recognition of transposon excision sites by thetransposase. In some embodiments, these excision sites may be terminalrepeats or inverted terminal repeats. Once excised from the transposon,the gene of interest can be integrated into the genome of a prokaryoticor eukaryotic cell by transposase-catalyzed cleavage of similar excisionsites that exist within nuclear genome of the cell. This allows the geneencoding an anti-TNFR2 antibody or fragment or domain thereof to beinserted into the cleaved nuclear DNA at the excision sites, andsubsequent ligation of the phosphodiester bonds that join the gene ofinterest to the DNA of the prokaryotic or eukaryotic cell genomecompletes the incorporation process. In some embodiments, the transposonmay be a retrotransposon, such that the gene encoding the antibody isfirst transcribed to an RNA product and then reverse-transcribed to DNAbefore incorporation in the prokaryotic or eukaryotic cell genome.Exemplary transposon systems include the piggyback transposon (describedin detail in WO 2010/085699) and the sleeping beauty transposon(described in detail in US20050112764); the disclosures of each of whichare incorporated herein by reference.

Another useful method for the integration of nucleic acid moleculesencoding anti-TNFR2 polypeptides (e.g., single-chain polypeptides,antibodies, or antigen-binding fragments thereof) described herein(e.g., any one or more of antibodies 1-25 described in Table 1 andvariants thereof, such as an antibody or antigen-binding fragmentcontaining one or more, or all, of the CDRs set forth in Table 1) intothe genome of a prokaryotic or eukaryotic cell is the clusteredregularly interspaced short palindromic repeats (CRISPR)/Cas system,which is a system that originally evolved as an adaptive defensemechanism in bacteria and archaea against infection by viruses. TheCRISPR/Cas system consists of palindromic repeat sequences withinplasmid DNA and an associated Cas9 nuclease. This ensemble of DNA andprotein directs site specific DNA cleavage of a target sequence by firstincorporating foreign DNA into CRISPR loci. Polynucleotides containingthese foreign sequences and the repeat-spacer elements of the CRISPRlocus are in turn transcribed in a host cell to create a guide RNA,which can subsequently anneal to a target sequence and localize the Cas9nuclease to this site. In this manner, highly site-specificcas9-mediated DNA cleavage can be engendered in a foreign polynucleotidebecause the interaction that brings cas9 within close proximity of thetarget DNA molecule is governed by RNA:DNA hybridization. As a result,one can theoretically design a CRISPR/Cas system to cleave any targetDNA molecule of interest. This technique has been exploited in order toedit eukaryotic genomes (Hwang et al., Nat. Biotech., 31:227-229, 2013)and can be used as an efficient means of site-specifically editingeukaryotic or prokaryotic genomes in order to cleave DNA prior to theincorporation of a polynucleotide encoding an anti-TNFR2 polypeptides(e.g., single-chain polypeptides, antibodies, or antigen-bindingfragments thereof) described herein. The use of CRISPR/Cas to modulategene expression has been described in U.S. Pat. No. 8,697,359, thedisclosure of which is incorporated herein by reference.

Alternative methods for site-specifically cleaving genomic DNA prior tothe incorporation of a polynucleotide encoding a TNFR2 antibody orantibody fragment described herein (e.g., any one or more of antibodies1-25 described in Table 1 and variants thereof, such as an antibody orantigen-binding fragment containing one or more, or all, of the CDRs setforth in Table 1) include the use of zinc finger nucleases andtranscription activator-like effector nucleases (TALENs). Unlike theCRISPR/Cas system, these enzymes do not contain a guiding polynucleotideto localize to a specific target sequence. Target specificity is insteadcontrolled by DNA binding domains within these enzymes. Zinc fingernucleases and TALENs for use in genome editing applications aredescribed in Urnov et al. (Nat. Rev. Genet., 11:636-646, 2010); and inJoung et al., (Nat. Rev. Mol. Cell. Bio. 14:49-55, 2013); incorporatedherein by reference. Additional genome editing techniques that can beused to incorporate polynucleotides encoding antibodies described hereininto the genome of a prokaryotic or eukaryotic cell include the use ofARCUS™ meganucleases that can be rationally designed so as tosite-specifically cleave genomic DNA. The use of these enzymes for theincorporation of polynucleotides encoding antagonistic TNFR2polypeptides (e.g., single-chain polypeptides, antibodies,antigen-binding fragments thereof, or constructs thereof) describedherein into the genome of a prokaryotic or eukaryotic cell isparticularly advantageous in view of the structure-activityrelationships that have been established for such enzymes. Single-chainmeganucleases can thus be modified at certain amino acid positions inorder to create nucleases that selectively cleave DNA at desiredlocations. These single-chain nucleases have been described extensively,e.g., in U.S. Pat. Nos. 8,021,867 and 8,445,251; the disclosures of eachof which are incorporated herein by reference.

Polynucleotide Sequence Elements

To express antagonistic TNFR2 polypeptides (e.g., single-chainpolypeptides, antibodies, antigen-binding fragments thereof, orconstructs thereof) described herein (e.g., any one or more ofantibodies 1-25 described in Table 1 and variants thereof, such as anantibody or antigen-binding fragment containing one or more, or all, ofthe CDRs set forth in Table 1), polynucleotides encoding partial orfull-length light and heavy chains, e.g., polynucleotides that encode aone or more, or all, of the CDR sequences of an antibody orantigen-binding fragment thereof described herein, can be inserted intoexpression vectors such that the genes are operatively linked totranscriptional and translational control sequences. The expressionvector and expression control sequences are chosen to be compatible withthe expression host cell used. Polynucleotides encoding the light chaingene and the heavy chain of a TNFR2 antibody can be inserted intoseparate vectors, or, optionally, both polynucleotides can beincorporated into the same expression vector using establishedtechniques described herein or known in the art.

In addition to polynucleotides encoding the heavy and light chains of anantibody (or a polynucleotide encoding a single-chain polypeptide, anantibody fragment, such as a scFv molecule, or a construct describedherein), the recombinant expression vectors described herein may carryregulatory sequences that control the expression of the antibody chaingenes in a host cell. The design of the expression vector, including theselection of regulatory sequences, may depend on such factors as thechoice of the host cell to be transformed or the level of expression ofprotein desired. For instance, suitable regulatory sequences formammalian host cell expression include viral elements that direct highlevels of protein expression in mammalian cells, such as promotersand/or enhancers derived from cytomegalovirus (CMV) (such as the CMVpromoter/enhancer), Simian Virus 40 (SV40) (such as the SV40promoter/enhancer), adenovirus, (e.g., the adenovirus major latepromoter (AdMLP)) and polyoma. Viral regulatory elements, and sequencesthereof, are described in detail, for instance, in U.S. Pat. Nos.5,168,062, 4,510,245, and 4,968,615, the disclosures of each of whichare incorporated herein by reference.

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors described herein can carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes. Aselectable marker gene facilitates selection of host cells into whichthe vector has been introduced (see e.g., U.S. Pat. Nos. 4,399,216,4,634,665 and 5,179,017). For example, typically the selectable markergene confers resistance to cytotoxic drugs, such as G418, puromycin,blasticidin, hygromycin or methotrexate, to a host cell into which thevector has been introduced. Suitable selectable marker genes include thedihydrofolate reductase (DHFR) gene (for use in DHFR″ host cells withmethotrexate selection/amplification) and the neo gene (for G418selection). In order to express the light and heavy chains of a TNFR2antibody or a TNFR2 antibody fragment, the expression vector(s)containing polynucleotides encoding the heavy and light chains can betransfected into a host cell by standard techniques.

Polynucleotides Encoding Modified Antagonistic TNFR2 Polypeptides

Antagonistic TNFR2 polypeptides of the disclosure include any one ormore of antibodies 1-25 described in Table 1 and variants thereof, suchas an antibody or antigen-binding fragment containing one or more, orall, of the CDRs set forth in Table 1. Additionally or alternatively,antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides,antibodies, antigen-binding fragments thereof, or constructs thereof)described herein may contain one or more, or all, of the CDRs ofTNFRAB1, TNFRAB2, TNFRAB3, TNFRAB4, or TNFRAB5, or one or more, or all,of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences inwhich one or more, or all, of the CDR sequences exhibits at least 85%sequence identity (e.g., 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity) to the corresponding CDR sequence of TNFRAB1, TNFRAB2,TNFRAB3, TNFRAB4, or TNFRAB5) or contains one or more (for instance, upto 3) amino acid substitutions (e.g., one or more conservative aminoacid substitutions) relative to the corresponding CDR sequence ofTNFRAB1, TNFRAB2, TNFRAB3, TNFRAB4, or TNFRAB5, but may featuredifferences in one or more framework regions of TNFRAB1, TNFRAB2,TNFRAB3, TNFRAB4, or TNFRAB5. For instance, one or more frameworkregions of TNFRAB1, TNFRAB2, TNFRAB3, TNFRAB4, or TNFRAB5 may besubstituted with the framework region of a human antibody. Exemplaryframework regions include, for example, human framework regionsdescribed in U.S. Pat. No. 7,829,086, and primate framework regions asdescribed in EP 1945668; the disclosures of each of which areincorporated herein by reference. To generate nucleic acids encodingsuch TNFR2 antibodies, DNA fragments encoding, e.g., at least one, orboth, of the light chain variable regions and the heavy chain variableregions can be produced by chemical synthesis (e.g., by solid phasepolynucleotide synthesis techniques), in vitro gene amplification (e.g.,by polymerase chain reaction techniques), or by replication of thepolynucleotide in a host organism. For instance, nucleic acids encodingantagonistic TNFR2 antibodies described herein may be obtained byamplification and modification of germline DNA or cDNA encoding lightand heavy chain variable sequences so as to incorporate one or more, orall, of the CDRs of TNFRAB1, TNFRAB2, TNFRAB3, TNFRAB4, or TNFRAB5 intothe framework residues of a consensus antibody.

In some embodiments, a humanized antagonistic TNFR2 antibody (such as ahumanized antibody described in Table 1) may include one or more, orall, of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequencesin which one or more, or all, of the CDR sequences exhibits at least 85%sequence identity (e.g., 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity) to the corresponding CDR sequence of TNFRAB1, TNFRAB2,TNFRAB3, TNFRAB4, or TNFRAB5) or contains one or more (for instance, upto 3) amino acid substitutions (e.g., one or more conservative aminoacid substitutions) relative to the corresponding CDR sequence ofTNFRAB1, TNFRAB2, TNFRAB3, TNFRAB4, or TNFRAB5. This can be achieved,for example, by performing site-directed mutagenesis of germline DNA orcDNA and amplifying the resulting polynucleotides using the polymerasechain reaction (PCR) according to established procedures. Germline DNAsequences for human heavy and light chain variable region genes areknown in the art (see, e.g., the “VBASE” human germline sequencedatabase; see also Kabat et al., Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242, 1991; Tomlinson et al., J. Mol. Biol.227:776-798, 1992; and Cox et al., Eur. J. Immunol. 24:827-836, 1994;incorporated herein by reference). Chimeric nucleic acid constructsencoding human heavy and light chain variable regions containing one ormore, or all, of the CDRs of TNFRAB1, TNFRAB2, TNFRAB3, TNFRAB4, orTNFRAB5, or a similar sequence as described above, can be produced,e.g., using established cloning techniques known in the art.Additionally, a polynucleotide encoding a heavy chain variable regioncontaining the one or more of the CDRs of TNFRAB1, TNFRAB2, TNFRAB3,TNFRAB4, or TNFRAB5, or a similar sequence as described above, can besynthesized and used as a template for mutagenesis to generate a variantas described herein using routine mutagenesis techniques. Alternatively,a DNA fragment encoding the variant can be directly synthesized (e.g.,by established solid phase nucleic acid chemical synthesis procedures).

Once DNA fragments encoding VH segments containing one or more, or all,of the CDR-H1, CDR-H2, and CDR-H3 sequences of TNFRAB1, TNFRAB2,TNFRAB3, TNFRAB4, or TNFRAB5 are obtained, these DNA fragments can befurther manipulated by standard recombinant DNA techniques, e.g., toconvert the variable region genes to full-length antibody chain genes,to Fab fragment genes or to a scFv gene. In these manipulations, a VL-or VH-encoding DNA fragment is operatively linked to another DNAfragment encoding another protein, such as an antibody constant regionor a flexible linker.

The isolated DNA encoding the VH region of an antagonistic TNFR2antibody described herein can be converted to a full-length heavy chaingene (as well as a Fab heavy chain gene), e.g., by operatively linkingthe V_(H)-encoding DNA to another DNA molecule encoding heavy chainconstant region domains (CH1, CH2, CH3, and, optionally, CH4). Thesequences of human heavy chain constant region genes are known in theart (see e.g., Kabat et al., Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242, 1991) and DNA fragments encompassing theseregions can be obtained by standard PCR amplification. The heavy chainconstant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgDconstant region, and in certain embodiments is an IgG1 constant region.For a Fab fragment heavy chain gene, the VH-encoding DNA can beoperatively linked to another DNA molecule encoding only the heavy chainCH1 domain.

Isolated DNA encoding the VL region of an antagonistic TNFR2 antibodycan be converted to a full-length light chain gene (as well as a Fablight chain gene) by operatively linking the VL-encoding DNA to anotherDNA molecule encoding the light chain constant region, CL. The sequencesof human light chain constant region genes are known in the art (seee.g., Kabat et al., Sequences of Proteins of Immunological Interest,Fifth Edition (U.S. Department of Health and Human Services, NIHPublication No. 91-3242, 1991)) and DNA fragments encompassing theseregions can be obtained, e.g., by amplification in a prokaryotic oreukaryotic cell of a polynucleotide encoding these regions, by PCRamplification, or by chemical polynucleotide synthesis. The light chainconstant region can be a kappa (κ) or lambda (λ) constant region, but incertain embodiments is a kappa constant region. To create a scFv gene,the VH and VL-encoding DNA fragments are operatively linked to anotherfragment encoding a flexible linker, e.g., a polynucleotide encoding aflexible, hydrophilic amino acid sequence, such as the amino acidsequence (Gly₄Ser)₃, such that the V_(H) and V_(L) sequences can beexpressed as a contiguous single-chain protein, with the V_(L) and V_(H)regions joined by the linker (see e.g., Bird et al., Science242:423-426, 1988; Huston et al., Proc. Natl. Acad. Sci. USA85:5879-5883, 1988; McCafferty et al., Nature 348:552-554, 1990).

Recombinant DNA technology can also be used to remove some or all of theDNA encoding either or both of the light and heavy chains that is notnecessary for binding to TNFR2. The molecules expressed from suchtruncated DNA molecules are also encompassed by the antibodies describedherein. In addition, bifunctional antibodies can be produced in whichone heavy contains one or more, or all, of the CDRs of TNFRAB1, TNFRAB2,TNFRAB3, TNFRAB4, or TNFRAB5, or a similar CDR sequence as describedabove, and the other heavy chain and/or the light chains are specificfor an antigen other than TNFR2. Such antibodies can be generated, e.g.,by crosslinking a heavy chain and light chain containing one or more, orall, of the CDRs of TNFRAB1, TNFRAB2, TNFRAB3, TNFRAB4, or TNFRAB5, or asimilar CDR sequence as described above, to a heavy chain and lightchain of a second antibody specific for a different antigen, forinstance, using standard chemical crosslinking methods (e.g., bydisulfide bond formation). Bifunctional antibodies can also be made byexpressing a nucleic acid molecule engineered to encode a bifunctionalantibody in a prokaryotic or eukaryotic cell.

Dual specific antibodies, i.e., antibodies that bind TNFR2 and adifferent antigen using the same binding site, can be produced bymutating amino acid residues in the light chain and/or heavy chain CDRs.In some embodiments, dual specific antibodies that bind two antigens,such as TNFR2 and a second cell-surface receptor, can be produced bymutating amino acid residues in the periphery of the antigen bindingsite (Bostrom et al., Science 323: 1610-1614, 2009). Dual functionalantibodies can be made by expressing a polynucleotide engineered toencode a dual specific antibody.

Modified antagonistic TNFR2 antibodies and antibody fragments describedherein can also be produced by chemical synthesis (e.g., by the methodsdescribed in Solid Phase Peptide Synthesis, 2nd ed., 1984 The PierceChemical Co., Rockford, 111; incorporated herein by reference). Variantantibodies can also be generated using a cell-free synthetic platform(see, e.g., Chu et al., Biochemia No. 2, 2001 (Roche MolecularBiologicals); incorporated herein by reference).

Host Cells for Expression of Antagonistic TNFR2 Polypeptides

It is possible to express the polypeptides (e.g., single-chainpolypeptides, antibodies, antigen-binding fragments thereof, andconstructs thereof) described herein (e.g., any one or more ofantibodies 1-25 described in Table 1 and variants thereof, such as anantibody or antigen-binding fragment containing one or more, or all, ofthe CDRs set forth in Table 1) in either prokaryotic or eukaryotic hostcells. In certain embodiments, expression of polypeptides (e.g.,single-chain polypeptides, antibodies, or antigen-binding fragmentsthereof) is performed in eukaryotic cells, e.g., mammalian host cells,for optimal secretion of a properly folded and immunologically activeantibody. Exemplary mammalian host cells for expressing the recombinantantibodies or antigen-binding fragments thereof described herein includeChinese Hamster Ovary (CHO cells) (including DHFR CHO cells, describedin Urlaub and Chasin (1980, Proc. Natl. Acad. Sci. USA 77:4216-4220),used with a DHFR selectable marker, e.g., as described in Kaufman andSharp (1982, Mol. Biol. 159:601-621), NSO myeloma cells, COS cells, 293cells, and SP2/0 cells. Additional cell types that may be useful for theexpression of antibodies and fragments thereof include bacterial cells,such as BL-21(DE3) E. coli cells, which can be transformed with vectorscontaining foreign DNA according to established protocols. Additionaleukaryotic cells that may be useful for expression of antibodies includeyeast cells, such as auxotrophic strains of S. cerevisiae, which can betransformed and selectively grown in incomplete media according toestablished procedures known in the art. When recombinant expressionvectors encoding antibody genes are introduced into mammalian hostcells, the antibodies are produced by culturing the host cells for aperiod of time sufficient to allow for expression of the antibody in thehost cells or secretion of the antibody into the culture medium in whichthe host cells are grown.

Polypeptides (e.g., single-chain polypeptides, antibodies,antigen-binding fragments thereof, and constructs thereof) can berecovered from the culture medium using standard protein purificationmethods. Host cells can also be used to produce portions of intactantibodies, such as Fab fragments or scFv molecules. Also includedherein are methods in which the above procedure is varied according toestablished protocols known in the art. For example, it can be desirableto transfect a host cell with DNA encoding either the light chain or theheavy chain (but not both) of an antagonistic TNFR2 antibody describedherein in order to produce an antigen-binding fragment of the antibody.

Once an antagonistic TNFR2 polypeptide (e.g., single-chain polypeptide,antibody, antigen-binding fragment thereof, or construct thereof)described herein (e.g., any one or more of antibodies 1-25 described inTable 1 and variants thereof, such as an antibody or antigen-bindingfragment containing one or more, or all, of the CDRs set forth inTable 1) has been produced by recombinant expression, it can be purifiedby any method known in the art, such as a method useful for purificationof an immunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for TNFR2 after Protein Aor Protein G selection, and sizing column chromatography),centrifugation, differential solubility, or by any other standardtechnique for the purification of proteins. Further, the antagonisticTNFR2 polypeptides described herein or fragments thereof can be fused toheterologous polypeptide sequences described herein or otherwise knownin the art to facilitate purification or to produce therapeuticconjugates (see “Antagonistic TNFR2 polypeptide conjugates,” below).

Once isolated, an anti-TNFR2 single-chain polypeptide, antibody, orantigen-binding fragments thereof can, if desired, be further purified,e.g., by high performance liquid chromatography (see, e.g., Fisher,Laboratory Techniques in Biochemistry and Molecular Biology (Work andBurdon, eds., Elsevier, 1980); incorporated herein by reference), or bygel filtration chromatography, such as on a Superdex™ 75 column(Pharmacia Biotech AB, Uppsala, Sweden).

Platforms for Generating and Affinity-Maturing Antagonistic Anti-TNFR2Polypeptides

Mapping Epitopes of TNFR2 that Promote Receptor Antagonism

Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides,antibodies, antigen-binding fragments thereof, and constructs thereof)described herein (e.g., any one or more of antibodies 1-25 described inTable 1 and variants thereof, such as an antibody or antigen-bindingfragment containing one or more, or all, of the CDRs set forth inTable 1) can be produced by screening libraries of polypeptides (e.g.,single-chain polypeptides, antibodies, antigen-binding fragmentsthereof, and constructs thereof) for functional molecules that arecapable of binding epitopes within TNFR2 that selectively promotereceptor antagonism rather than receptor activation. Such epitopes canbe modeled by screening antibodies or antigen-binding fragments thereofagainst a series of linear or cyclic peptides containing residues thatcorrespond to a desired epitope within TNFR2.

As an example, peptides containing individual fragments isolated fromTNFR2 that promote receptor antagonism can be synthesized by peptidesynthesis techniques described herein or known in the art. Thesepeptides can be immobilized on a solid surface and screened formolecules that bind antagonistic TNFR2 polypeptides (e.g., single-chainpolypeptides, antibodies, antigen-binding fragments thereof, andconstructs thereof), e.g., using an ELISA-based screening platform usingestablished procedures. Using this assay, peptides that specificallybind TNFRAB1, TNFRAB2, TNFRAB3, TNFRAB4, or TNFRAB5, or any one or moreof antibodies 1-25 described in Table 1 or a variant thereof, such as anantibody or antigen-binding fragment containing one or more, or all, ofthe CDRs set forth in Table 1, with high affinity therefore containresidues within epitopes of TNFR2 that preferentially bind theseantibodies. Peptides identified in this manner (e.g., peptides havingthe sequence of any one of SEQ ID NOs: 11, 19, 20, and 34-117) can beused to screen libraries of polypeptides (e.g., single-chainpolypeptides, antibodies, antigen-binding fragments thereof, andconstructs thereof) in order to identify antagonistic TNFR2polypeptides. Moreover, since these peptides act as surrogates forepitopes within TNFR2 that promote receptor antagonism, polypeptidesgenerated using this screening technique may bind the correspondingepitopes in TNFR2 and are expected to be antagonistic of receptoractivity.

Screening of Libraries for Antagonistic TNFR2 Polypeptides

Methods for high throughput screening of polypeptide (e.g., single-chainpolypeptide, antibody, antibody fragment, or construct thereof)libraries for molecules capable of binding epitopes within TNFR2 (e.g.,peptides having the sequence of any one of SEQ ID NOs: 11, 19, 20, and34-117) include, without limitation, display techniques including phagedisplay, bacterial display, yeast display, mammalian display, ribosomedisplay, mRNA display, and cDNA display. The use of phage display toisolate ligands that bind biologically relevant molecules has beenreviewed, e.g., in Felici et al. (Biotechnol. Annual Rev. 1:149-183,1995), Katz (Annual Rev. Biophys. Biomol. Struct. 26:27-45, 1997), andHoogenboom et al. (Immunotechnology 4:1-20, 1998). Several randomizedcombinatorial peptide libraries have been constructed to select forpolypeptides that bind different targets, e.g., cell surface receptorsor DNA (reviewed by Kay (Perspect. Drug Discovery Des. 2, 251-268,1995), Kay et al., (Mol. Divers. 1:139-140, 1996)). Proteins andmultimeric proteins have been successfully phage-displayed as functionalmolecules (see EP 0349578A, EP 4527839A, EP 0589877A; Chiswell andMcCafferty (Trends Biotechnol. 10, 80-84 1992)). In addition, functionalantibody fragments (e.g. Fab, single-chain Fv [scFv]) have beenexpressed (McCafferty et al. (Nature 348: 552-554, 1990), Barbas et al.(Proc. Natl. Acad Sci. USA 88:7978-7982, 1991), Clackson et al. (Nature352:624-628, 1991)). These references are hereby incorporated byreference in their entirety.

(i) Phage Display Techniques

As an example, phage display techniques can be used in order to screenlibraries of polypeptides (e.g., single-chain polypeptides, antibodies,antigen-binding fragments thereof, and constructs thereof) forfunctional molecules capable of binding cyclic or polycyclic peptidescontaining epitopes within TNFR2 that promote receptor antagonism (e.g.,peptides having the sequence of any one of SEQ ID NOs: 11, 19, 20, and34-117). For instance, libraries of polynucleotides encodingsingle-chain antibody fragments, such as scFv fragments, that containrandomized hypervariable regions can be obtained using establishedprocedures (e.g., solid phase polynucleotide synthesis or error-pronePCR techniques, see McCullum et al. (Meth. Mol. Biol., 634:103-109,2010); incorporated herein by reference). These randomizedpolynucleotides can subsequently be incorporated into a viral genomesuch that the randomized antibody chains encoded by these genes areexpressed on the surface of filamentous phage, e.g., by a covalent bondbetween the antibody chain and a coat protein (e.g., pill coat proteinon the surface of M13 phage). This provides a physical connectionbetween the genotype and phenotype of the antibody chain. In this way,libraries of phage that display diverse antibody chains containingrandom mutations in hypervariable regions can be screened for theability of the exterior antibody chains to bind TNFR2 epitopes (e.g.,peptides having the sequence of any one of SEQ ID NOs: 11, 19, 20, and34-117) that are immobilized to a surface using established procedures.For instance, such peptides can be physically bound to the surface of amicrotiter plate by forming a covalent bond between the peptide and anepitope tag (e.g., biotin) and incubating the peptide in wells of amicrotiter plate that have been previously coated with a complementarytag (e.g., avidin) that binds the tag attached to the peptide with highaffinity. Suitable epitope tags include, without limitation,maltose-binding protein, glutathione-S-transferase, a poly-histidinetag, a FLAG-tag, a myc-tag, human influenza hemagglutinin (HA) tag,biotin, streptavidin. Peptides containing the epitopes presented bythese molecules are capable of being immobilized on surfaces containingsuch complementary molecules as maltose, glutathione, anickel-containing complex, an anti-FLAG antibody, an anti-myc antibody,an anti-HA antibody, streptavidin, or biotin, respectively. In this way,phage can be incubated with a surface containing an immobilizedTNFR2-derived peptide for a time suitable to allow binding of theantibody to the constrained peptide and in the presence of anappropriate buffer system (e.g., one that contains physiological saltconcentration, ionic strength, and is maintained at physiological pH bya buffering agent). The surface can then be washed (e.g., with phosphatebuffer containing 0.1% Tween-20) so as to remove phage that do notpresent antibody chains that interact with the TNFR2-derived peptideswith an affinity greater than a particular threshold value.

The affinity of the polypeptides that remain after this initial panning(i.e., screening) step can be modulated by adjusting the conditions ofthe washing step (e.g., by including mildly acidic or basic components,or by including other TNFR2-derived peptides at a low concentration inorder to compete with immobilized peptides for antigen-binding sites).In this way, the population of phage that remains bound to the surfacesof the microtiter plate following the washing step is enriched for phagethat bind TNFR2-derived peptide epitopes that promote receptorantagonism. The remaining phage can then be amplified by eluting thephage from the surface containing these peptides (e.g., by altering theambient pH, ionic strength, or temperature) so as to diminishprotein-protein interaction strength. The isolated phage can then beamplified, e.g., by infecting bacterial cells, and the resulting phagecan optionally be subjected to panning by additional iterations ofscreening so as to further enrich the population of phage for thoseharboring higher-affinity anti-TNFR2 polypeptides. Following thesepanning stages, phage that display high-affinity antibodies orantigen-binding fragments thereof can subsequently be isolated and thegenomes of these phage can be sequenced in order to identify thepolynucleotide and polypeptide sequences of the encoded antibodies.Phage display techniques such as this can be used to generate, e.g.,antibody chains, such as scFv fragments, tandem scFv fragments, andother antigen-binding fragments described herein that can be used asantagonists of TNFR2. Exemplary phage display protocols for theidentification of antibody chains and antigen-binding fragments thereofthat bind a particular antigen with high affinity are well-establishedand are described, e.g., in U.S. Pat. No. 7,846,892, WO 1997/002342,U.S. Pat. No. 8,846,867, and WO 2007/132917; the disclosures of each ofwhich are incorporated herein by reference. Similar phage displaytechniques can be used to generate antibody-like scaffolds (e.g., ¹⁰Fn3domains) described herein that bind epitopes within TNFR2 that promotereceptor antagonism (e.g., epitopes presented by peptides with thesequence of any one of SEQ ID NOs: 11, 19, 20, and 34-117). Exemplaryphage display protocols for the identification of antibody-like scaffoldproteins are described, e.g., in WO 2009/086116; the disclosure of whichis incorporated herein by reference).

(ii) Cell-Based Display Techniques

Other in vitro display techniques that exploit the linkage betweengenotype and phenotype of a solvent-exposed polypeptide include yeastand bacterial display. Yeast display techniques are established in theart and are often advantageous in that high quantities of antibodies(often up to 30,000) can be presented on the surface of an individualyeast cell (see, e.g., Boder et al. (Nat Biotechno. 15:553, 1997);incorporated herein by reference). The larger size of yeast cells overfilamentous phage enables an additional screening strategy, as one canuse flow cytometry to both analyze and sort libraries of yeast. Forinstance, established procedures can be used to generate libraries ofbacterial cells or yeast cells that express polypeptides (e.g.,single-chain polypeptides, antibodies, and antigen-binding fragmentsthereof) containing randomized hypervariable regions (see, e.g., seeU.S. Pat. No. 7,749,501 and US 2013/0085072; the teachings of each whichare incorporated herein by reference). For instance, large libraries ofyeast cells that express polynucleotides encoding naïve scFv fragmentscan be made using established procedures (de Bruin et al., NatBiotechnol 17:397, 1999; incorporated herein by reference). Yeast cellsexpressing these polynucleotides can then be incubated with twodifferent fluorescent molecules during the panning steps: one dye thatbinds conserved residues within the antibody and thus reflects theamount of antibody displayed, and another dye that fluoresces at adifferent wavelength and binds the antigen and thus indicates the amountof antigen bound. For instance, one of skill in the art can use aTNFR2-derived peptide containing the sequence of any one of SEQ ID NOs:11, 19, 20, and 34-117 that has been conjugated to an epitope tag (e.g.,biotin), optionally at the N- or C-terminus of the peptide or at aresidue that is not expected to interfere with antibody-antigen binding.This enables a fluorescent dye labeled with a complementary tag (e.g.,avidin) to localize to the antibody-antigen complex. This results ingreat flexibility and immediate feedback on the progress of a selection.In contrast to phage display, by normalizing to antibody display levels,antibodies with higher affinities, rather than greater expression levelscan easily be selected. In fact, it is possible to distinguish and sortantibodies whose affinities differ by only two-fold (VanAntwerp andWittrup (Biotechnol Prog 16:31, 2000)).

(iii) Nucleotide Display Techniques

Display techniques that utilize in vitro translation of randomizedpolynucleotide libraries also provide a powerful approach to generatingantagonistic TNFR2 polypeptides described herein. For instance,randomized DNA libraries encoding polypeptides (e.g., single-chainpolypeptides, antibodies, and antigen-binding fragments thereof) thatcontain mutations within designated hypervariable regions can beobtained, e.g., using established PCR-based mutagenesis techniques asdescribed herein. The polynucleotides of these libraries may containtranscription regulating sequences, such as promoters and transcriptionterminating sequences, and may additionally encode sequences thatincrease the rate of translation of the resulting mRNA construct (e.g.,IRES sequences, 5′ and 3′ UTRs, a poly-adenylation tract, etc). Thesepolynucleotide libraries can be incubated in an appropriately bufferedsolution containing RNA polymerase and RNA nucleoside triphosphates(NTPs) in order to enable transcription of the DNA sequences tocompetent mRNA molecules, which can subsequently be translated by largeand small ribosomal subunits, aminoacyl tRNA molecules, and translationinitiation and elongation factors present in solution (e.g., using thePURExpress® In Vitro Protein Synthesis Kit, New England Biolabs®).Designed mRNA modifications can enable the antibody product to remaincovalently bound to the mRNA template by a chemical bond to puromycin(e.g., see Keefe (Curr. Protoc. Mol. Biol., Chapter 24, Unit 24.5,2001); incorporated herein by reference). This genotype-phenotypelinkage can thus be used to select for antibodies that bind aTNFR2-derived peptide (e.g., a peptide that has the sequence of any oneof SEQ ID NOs: 11, 19, 20, and 34-117) by incubating mRNA:antibodyfusion constructs with a peptide immobilized to a surface and panning ina fashion similar to phage display techniques (see, e.g., WO2006/072773; incorporated herein by reference).

Optionally, polypeptides (e.g., single-chain polypeptides, antibodies,and antigen-binding fragments thereof) described herein can be generatedusing a similar technique, except the antibody product may be boundnon-covalently to the ribosome-mRNA complex rather than covalently via apuromycin linker. This platform, known as ribosome display, has beendescribed, e.g., in U.S. Pat. No. 7,074,557; incorporated herein byreference. Alternatively, antibodies can be generated using cDNAdisplay, a technique analogous to mRNA display with the exception thatcDNA, rather than mRNA, is covalently bound to an antibody product via apuromycin linker. cDNA display techniques offer the advantage of beingable to perform panning steps under increasingly stringent conditions,e.g., under conditions in which the salt concentration, ionic strength,pH, and/or temperature of the environment is adjusted in order to screenfor antibodies with particularly high affinity for TNFR2-derivedpeptides. This is due to the higher natural stability of double-strandedcDNA over single-stranded mRNA. cDNA display screening techniques aredescribed, e.g., in Ueno et al. (Methods Mol. Biol., 805:113-135, 2012);incorporated herein by reference.

In addition to generating antagonistic TNFR2 polypeptides (e.g.,single-chain polypeptides, antibodies, antigen-binding fragmentsthereof, and constructs thereof) described herein, in vitro displaytechniques (e.g., those described herein and those known in the art)also provide methods for improving the affinity of an antagonistic TNFR2polypeptide described herein. For instance, rather than screeninglibraries of antibodies and fragments thereof containing completelyrandomized hypervariable regions, one can screen narrower libraries ofantibodies and antigen-binding fragments thereof that feature targetedmutations at specific sites within hypervariable regions. This can beaccomplished, e.g., by assembling libraries of polynucleotides encodingantibodies or antigen-binding fragments thereof that encode randommutations only at particular sites within hypervariable regions. Thesepolynucleotides can then be expressed in, e.g., filamentous phage,bacterial cells, yeast cells, mammalian cells, or in vitro using, e.g.,ribosome display, mRNA display, or cDNA display techniques in order toscreen for antibodies or antigen-binding fragments thereof thatspecifically bind TNFR2 epitopes (e.g., peptides containing the sequenceof any one of SEQ ID NOs: 11, 19, 20, and 34-117) with improved bindingaffinity. Yeast display, for instance, is well-suited for affinitymaturation, and has been used previously to improve the affinity of asingle-chain antibody to a K_(D) of 48 fM (Boder et al. (Proc Natl AcadSci USA 97:10701, 2000)).

Additional in vitro techniques that can be used for the generation andaffinity maturation of antagonistic TNFR2 polypeptides (e.g.,single-chain polypeptides, antibodies, and antigen-binding fragmentsthereof) described herein include the screening of combinatoriallibraries of antibodies or antigen-binding fragments thereof forfunctional molecules capable of specifically binding TNFR2-derivedpeptides (e.g., a peptide having the amino acid sequence of any one ofSEQ ID NOs: 11, 19, 20, and 34-117). Combinatorial antibody librariescan be obtained, e.g., by expression of polynucleotides encodingrandomized hypervariable regions of an antibody or antigen-bindingfragment thereof in a eukaryotic or prokaryotic cell. This can beachieved, e.g., using gene expression techniques described herein orknown in the art. Heterogeneous mixtures of antibodies can be purified,e.g., by Protein A or Protein G selection, sizing columnchromatography), centrifugation, differential solubility, and/or by anyother standard technique for the purification of proteins. Libraries ofcombinatorial libraries thus obtained can be screened, e.g., byincubating a heterogeneous mixture of these antibodies with a peptidederived from TNFR2 that has been immobilized to a surface (e.g., apeptide having the amino acid sequence of any one of SEQ ID NOs: 11, 19,20, and 34-117 immobilized to the surface of a solid-phase resin or awell of a microtiter plate) for a period of time sufficient to allowantibody-antigen binding. Non-binding antibodies or fragments thereofcan be removed by washing the surface with an appropriate buffer (e.g.,a solution buffered at physiological pH (approximately 7.4) andcontaining physiological salt concentrations and ionic strength, andoptionally containing a detergent, such as TWEEN-20). Antibodies thatremain bound can subsequently be detected, e.g., using an ELISA-baseddetection protocol (see, e.g., U.S. Pat. No. 4,661,445; the disclosureof which is incorporated herein by reference).

Additional techniques for screening combinatorial libraries ofpolypeptides (e.g., single-chain polypeptides, antibodies,antigen-binding fragments thereof, and constructs thereof) for thosethat specifically bind TNFR2-derived peptides (e.g., a peptidecontaining the amino acid sequence of any one of SEQ ID NOs: 11, 19, 20,and 34-117) include the screening of one-bead-one-compound libraries ofantibody fragments. Antibody fragments can be chemically synthesized ona solid bead (e.g., using established split-and-pool solid phase peptidesynthesis protocols) composed of a hydrophilic, water-swellable materialsuch that each bead displays a single antibody fragment. Heterogeneousbead mixtures can then be incubated with a TNFR2-derived peptide that isoptionally labeled with a detectable moiety (e.g., a fluorescent dye) orthat is conjugated to an epitope tag (e.g., biotin, avidin, FLAG tag, HAtag) that can later be detected by treatment with a complementary tag(e.g., avidin, biotin, anti-FLAG antibody, anti-HA antibody,respectively). Beads containing antibody fragments that specificallybind a TNFR2-derived peptide (e.g., a peptide containing the amino acidsequence of any one of SEQ ID NOs: 11, 19, 20, and 34-117) can beidentified by analyzing the fluorescent properties of the beadsfollowing incubation with a fluorescently-labeled antigen orcomplementary tag (e.g., by confocal fluorescent microscopy or byfluorescence-activated bead sorting; see, e.g., Muller et al. (J. Biol.Chem., 16500-16505, 1996); incorporated herein by reference). Beadscontaining antibody fragments that specifically bind TNFR2-derivedpeptides can thus be separated from those that do not containhigh-affinity antibody fragments. The sequence of an antibody fragmentthat specifically binds a TNFR2-derived peptide can be determined bytechniques known in the art, including, e.g., Edman degradation, tandemmass spectrometry, matrix-assisted laser-desorption time-of-flight massspectrometry (MALDI-TOF MS), nuclear magnetic resonance (NMR), and 2Dgel electrophoresis, among others (see, e.g., WO 2004/062553; thedisclosures of each of which are incorporated herein by reference).

Negative Screens of Polypeptides

In addition to the above-described methods for screening for asingle-chain polypeptide, antibody, or antibody fragment thatspecifically binds to an epitope derived from human TNFR2 that promotesreceptor antagonism, one can additionally perform negative screens inorder to eliminate antibodies or antibody fragments that may also bindan epitope that contains the KCSPG sequence. For instance, mixtures ofantibodies or antibody fragments isolated as a result of any of theabove-described screening techniques can be screened for antibodies orantibody fragments that also specifically bind to a peptide derived fromhuman TNFR2 that contains the KCSPG motif, such as a peptide containingresidues 48-67 of SEQ ID NO: 7 (QTAQMCCSKCSPGQHAKVFC, SEQ ID NO: 18).This can be accomplished using any of the above-described methods orvariations thereof, e.g., such that the antibodies or antibody fragmentsbeing screened are those that were previously identified as beingcapable of specifically binding a peptide containing one or moreresidues of SEQ ID NOs: 11, 19, 20, and 34-117. Exemplary techniquesuseful for a negative screen include those described above or known inthe art, such as phage display, yeast display, bacterial display,ribosome display, mRNA display, cDNA display, or surface-basedcombinatorial library screens (e.g., in an ELISA format). This screeningtechnique represents a useful strategy for identifying an antagonisticTNFR2 antibody or antibody fragment, as antibodies or antibody fragmentscapable of binding TNFR2 epitopes containing the KCSPG sequence lack, orhave significantly reduced, antagonistic activity.

Immunization of a Non-Human Mammal

Another strategy that can be used to produce antagonistic TNFR2antibodies and antigen-binding fragments thereof described hereinincludes immunizing a non-human mammal. Examples of non-human mammalsthat can be immunized in order to produce antagonistic TNFR2 antibodiesand fragments thereof described herein include rabbits, mice, rats,goats, guinea pigs, hamsters, horses, and sheep, as well as non-humanprimates. For instance, established procedures for immunizing primatesare known in the art (see, e.g., WO 1986/6004782; incorporated herein byreference). Immunization represents a robust method of producingmonoclonal antibodies by exploiting the antigen specificity of Blymphocytes. For example, monoclonal antibodies can be prepared by theKohler-Millstein procedure (described, e.g., in EP 0110716; incorporatedherein by reference), wherein spleen cells from a non-human animal(e.g., a primate) immunized with a peptide that presents a TNFR2-derivedantigen that promotes receptor antagonism (e.g., a peptide containingthe amino acid sequence of any one of SEQ ID NOs: 11, 19, 20, and34-117). A clonally-expanded B lymphocyte produced by immunization canbe isolated from the serum of the animal and subsequently fused with amyeloma cell in order to form a hybridoma. Hybridomas are particularlyuseful agents for antibody production, as these immortalized cells canprovide a lasting supply of an antigen-specific antibody. Antibodiesfrom such hybridomas can subsequently be isolated using techniques knownin the art, e.g., by purifying the antibodies from the cell culturemedium by affinity chromatography, using reagents such as Protein A orProtein G.

Antagonistic TNFR2 Polypeptide Conjugates

Prior to administration of antagonistic TNFR2 polypeptides (e.g.,single-chain polypeptides, antibodies, and antigen-binding fragmentsthereof) described herein (e.g., any one or more of antibodies 1-25described in Table 1 and variants thereof, such as an antibody orantigen-binding fragment containing one or more, or all, of the CDRs setforth in Table 1) to a mammalian subject (e.g., a human), it may bedesirable to conjugate the antibody or fragment thereof to a secondmolecule, e g., to modulate the activity of the antibody in vivo.Antagonistic TNFR2 antibodies and fragments thereof can be conjugated toother molecules at either the N-terminus or C-terminus of a light orheavy chain of the antibody using any one of a variety of establishedconjugation strategies that are well-known in the art. Examples of pairsof reactive functional groups that can be used to covalently tether anantagonistic TNFR2 antibody or fragment thereof to another moleculeinclude, without limitation, thiol pairs, carboxylic acids and aminogroups, ketones and amino groups, aldehydes and amino groups, thiols andalpha,beta-unsaturated moieties (such as maleimides or dehydroalanine),thiols and alpha-halo amides, carboxylic acids and hydrazides, aldehydesand hydrazides, and ketones and hydrazides.

Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides,antibodies, antigen-binding fragments thereof, and constructs thereof)can be covalently appended directly to another molecule by chemicalconjugation as described. Alternatively, fusion proteins containingantagonistic TNFR2 antibodies and fragments thereof can be expressedrecombinantly from a cell (e.g., a eukaryotic cell or prokaryotic cell).This can be accomplished, for example, by incorporating a polynucleotideencoding the fusion protein into the nuclear genome of a cell (e.g.,using techniques described herein or known in the art). Optionally,antibodies and fragments thereof described herein can be joined to asecond molecule by forming a covalent bond between the antibody and alinker. This linker can then be subsequently conjugated to anothermolecule, or the linker can be conjugated to another molecule prior toligation to the anti-TNFR2 antibody or fragment thereof. Examples oflinkers that can be used for the formation of a conjugate includepolypeptide linkers, such as those that contain naturally occurring ornon-naturally occurring amino acids. In some embodiments, it may bedesirable to include D-amino acids in the linker, as these residues arenot present in naturally-occurring proteins and are thus more resistantto degradation by endogenous proteases. Fusion proteins containingpolypeptide linkers can be made using chemical synthesis techniques,such as those described herein, or through recombinant expression of apolynucleotide encoding the fusion protein in a cell (e.g., aprokaryotic or eukaryotic cell). Linkers can be prepared using a varietyof strategies that are well known in the art, and depending on thereactive components of the linker, can be cleaved by enzymatichydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysisunder basic conditions, oxidation, disulfide reduction, nucleophiliccleavage, or organometallic cleavage (Leriche et al., Bioorg. Med.Chem., 20:571-582, 2012).

Drug-Polypeptide Conjugates

An antagonistic TNFR2 polypeptide (e.g., single-chain polypeptide,antibody, and antigen-binding fragment thereof) described herein (e.g.,any one or more of antibodies 1-25 described in Table 1 and variantsthereof, such as an antibody or antigen-binding fragment containing oneor more, or all, of the CDRs set forth in Table 1) can additionally beconjugated to, admixed with, or administered separately from atherapeutic agent, such as a cytotoxic molecule. Conjugates describedherein may be applicable to the treatment or prevention of a diseaseassociated with aberrant cell proliferation, such as a cancer describedherein. Exemplary cytotoxic agents that can be conjugated to, admixedwith, or administered separately from an antagonistic TNFR2 polypeptideinclude, without limitation, antineoplastic agents such as: acivicin;aclarubicin; acodazole hydrochloride; acronine; adozelesin; adriamycin;aldesleukin; altretamine; ambomycin; a. metantrone acetate;aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase;asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa;bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin;bleomycin sulfate; brequinar sodium; bropirimine; busulfan;cactinomycin; calusterone; camptothecin; caracemide; carbetimer;carboplatin; carmustine; carubicin hydrochloride; carzelesin;cedefingol; chlorambucil; cirolemycin; cisplatin; cladribine;combretestatin a-4; crisnatol mesylate; cyclophosphamide; cytarabine;dacarbazine; daca (n-[2-(dimethyl-amino) ethyl] acridine-4-carboxamide);dactinomycin; daunorubicin hydrochloride; daunomycin; decitabine;dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel;dolastatins; doxorubicin; doxorubicin hydrochloride; droloxifene;droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate;eflornithine hydrochloride; ellipticine; elsamitrucin; enloplatin;enpromate; epipropidine; epirubicin hydrochloride; erbulozole;esorubicin hydrochloride; estramustine; estramustine phosphate sodium;etanidazole; ethiodized oil i 131; etoposide; etoposide phosphate;etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine;fludarabine phosphate; fluorouracil; 5-fdump; flurocitabine; fosquidone;fostriecin sodium; gemcitabine; gemcitabine hydrochloride; gold au 198;homocamptothecin; hydroxyurea; idarubicin hydrochloride; ifosfamide;ilmofosine; interferon alfa-2a; interferon alfa-2b; interferon alfa-nI;interferon alfa-n3; interferon beta-i a; interferon gamma-i b;iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole;leuprolide acetate; liarozole hydrochloride; lometrexol sodium;lomustine; losoxantrone hydrochloride; masoprocol; maytansine;mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate;melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium;metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin;mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride;mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran;paclitaxel; pegaspargase; peliomycin; pentamustine; peploycinsulfate;perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride;plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine;procarbazine hydrochloride; puromycin; puromycin hydrochloride;pyrazofurin; rhizoxin; rhizoxin d; riboprine; rogletimide; safingol;safingol hydrochloride; semustine; simtrazene; sparfosate sodium;sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin;streptonigrin; streptozocin; strontium chloride sr 89; sulofenur;talisomycin; taxane; taxoid; tecogalan sodium; tegafur; teloxantronehydrochloride; temoporfin; teniposide; teroxirone; testolactone;thiamiprine; thioguanine; thiotepa; thymitaq; tiazofurin; tirapazamine;tomudex; top53; topotecan hydrochloride; toremifene citrate; trestoloneacetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate;triptorelin; tubulozole hydrochloride; uracil mustard; uredepa;vapreotide; verteporfin; vinblastine; vinblastine sulfate; vincristine;vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate;vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate;vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin;zinostatin; zorubicin hydrochloride; 2-chlorodeoxyadenosine; 2′deoxyformycin; 9-aminocamptothecin; raltitrexed;N-propargyl-5,8-dideazafolic acid;2chloro-2′-arabino-fluoro-2′-deoxyadenosine; 2-chloro-2′-deoxyadenosine;anisomycin; trichostatin A; hPRL-G129R; CEP-751; linomide; sulfurmustard; nitrogen mustard (mechlor ethamine); cyclophosphamide;melphalan; chlorambucil; ifosfamide; busulfan; N-methyl-Nnitrosourea(MNU); N, N′-Bis (2-chloroethyl)-N-nitrosourea (BCNU);N-(2-chloroethyl)-N′ cyclohexyl-N-nitrosourea (CCNU);N-(2-chloroethyl)-N′-(trans-4-methylcyclohexyl-N-nitrosourea (MeCCNU);N-(2-chloroethyl)-N′-(diethyl) ethylphosphonate-N-nitrosourea(fotemustine); streptozotocin; diacarbazine (DTIC); mitozolomide;temozolomide; thiotepa; mitomycin C; AZQ; adozelesin; cisplatin;carboplatin; ormaplatin; oxaliplatin; C1-973; DWA 2114R; JM216; JM335;Bis (platinum); tomudex; azacitidine; cytarabine; gemcitabine;6-mercaptopurine; 6-thioguanine; hypoxanthine; teniposide 9-aminocamptothecin; topotecan; CPT-11; Doxorubicin; Daunomycin; Epirubicin;darubicin; mitoxantrone; losoxantrone; Dactinomycin (Actinomycin D);amsacrine; pyrazoloacridine; all-trans retinol;14-hydroxy-retro-retinol; all-trans retinoic acid; N-(4-hydroxyphenyl)retinamide; 13-cis retinoic acid; 3-methyl TTNEB; 9-cis retinoic acid;fludarabine (2-F-ara-AMP); or 2-chlorodeoxyadenosine (2-Cda).

Other therapeutic compounds that can be conjugated to, admixed with, oradministered separately from an antagonistic TNFR2 single-chainpolypeptide, antibody, or antigen-binding fragment thereof describedherein in order to treat, prevent, or study the progression of a diseaseassociated with aberrant cell proliferation include, but are not limitedto, cytotoxic agents such as 20-pi-1,25 dihydroxyvitamin D3;5-ethynyluracil; abiraterone; acylfulvene; adecypenol; adozelesin;aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox;amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide;anastrozole; andrographolide; angiogenesis inhibitors; antagonist D;antagonist G; antarelix; anti-dorsalizing morphogenetic protein-1;antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston;antisense oligonucleotides; aphidicolin glycinate; apoptosis genemodulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA;argininedeaminase; asulacrine; atamestane; atrimustine; axinastatin 1;axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatinIII derivatives; balanol; batimastat; BCR/ABL antagonists;benzochlorins; benzoylstaurosporine; beta lactam derivatives;beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor;bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistrateneA; bizelesin; breflate; bleomycin A2; bleomycin B2; bropirimine;budotitane; buthionine sulfoximine; calcipotriol; calphostin C;camptothecin derivatives (e.g., 10-hydroxy-camptothecin); canarypoxIL-2; capecitabine; carboxamide-amino-triazole; carboxyamidotriazole;CaRest M3; CARN 700; cartilage derived inhibitor; carzelesin; caseinkinase inhibitors (ICOS); castanospermine; cecropin B; cetrorelix;chlorins; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin;cladribine; clomiphene analogues; clotrimazole; collismycin A;collismycin B; combretastatin A4; combretastatin analogue; conagenin;crambescidin 816; crisnatol; cryptophycin 8; cryptophycin A derivatives;curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabineocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine;dehydrodidemnin B; 2′deoxycoformycin (DCF); deslorelin; dexifosfamide;dexrazoxane; dexverapamil; diaziquone; didemnin B; didox;diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin;diphenyl spiromustine; discodermolide; docosanol; dolasetron;doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen;ecomustine; edelfosine; edrecolomab; eflornithine; elemene; emitefur;epirubicin; epothilones (A, R=H; B, R=Me); epothilones; epristeride;estramustine analogue; estrogen agonists; estrogen antagonists;etanidazole; etoposide; etoposide 4′-phosphate (etopofos); exemestane;fadrozole; fazarabine; fenretinide; filgrastim; finasteride;flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicinhydrochloride; forfenimex; formestane; fostriecin; fotemustine;gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix;gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam;heregulin; hexamethylene bisacetamide; homoharringtonine (HHT);hypericin; ibandronic acid; idarubicin; idoxifene; idramantone;ilmofosine; ilomastat; imidazoacridones; imiquimod; immunostimulantpeptides; insulin-like growth factor-1 receptor inhibitor; interferonagonists; interferons; interleukins; iobenguane; iododoxorubicin;ipomeanol; irinotecan; iroplact; irsogladine; isobengazole;isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F;lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinansulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocytealpha interferon; leuprolide+estrogen+progesterone; leuprorelin;levamisole; liarozole; linear polyamine analogue; lipophilicdisaccharide peptide; lipophilic platinum compounds; lissoclinamide 7;lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone;lovastatin; loxoribine; lurtotecan; lutetium texaphyrin; lysofylline;lytic peptides; maytansine; mannostatin A; marimastat; masoprocol;maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors;menogaril; rnerbarone; meterelin; methioninase; metoclopramide; MIFinhibitor; ifepristone; miltefosine; mirimostim; mismatched doublestranded RNA; mithracin; mitoguazone; mitolactol; mitomycin analogues;mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone;mofarotene; molgramostim; monoclonal antibody, human chorionicgonadotrophin; monophosphoryl lipid A+myobacterium cell wall sk;mopidamol; multiple drug resistance gene inhibitor; multiple tumorsuppressor 1-based therapy; mustard anticancer agent; mycaperoxide B;mycobacterial cell wall extract; myriaporone; N-acetyldinaline;N-substituted benzamides; nafarelin; nagrestip; naloxone+pentazocine;napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronicacid; neutral endopeptidase; nilutamide; nisamycin; nitric oxidemodulators; nitroxide antioxidant; nitrullyn; 06-benzylguanine;octreotide; okicenone; oligonucleotides; onapristone; ondansetron;ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone;oxaliplatin; oxaunomycin; paclitaxel analogues; paclitaxel derivatives;palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene;parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfatesodium; pentostatin; pentrozole; perflubron; perfosfamide; perillylalcohol; phenazinomycin; phenylacetate; phosphatase inhibitors;picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetinA; placetin B; plasminogen activator inhibitor; platinum complex;platinum compounds; platinum-triamine complex; podophyllotoxin; porfimersodium; porfiromycin; propyl bis-acridone; prostaglandin J2; proteasomeinhibitors; protein A-based immune modulator; protein kinase Cinhibitor; protein kinase C inhibitors, microalgal; protein tyrosinephosphatase inhibitors; purine nucleoside phosphorylase inhibitors;purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethyleneconjugate; raf antagonists; raltitrexed; ramosetron; ras farnesylprotein transferase inhibitors; ras inhibitors; ras-GAP inhibitor;retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin;ribozymes; RII retinamide; rogletimide; rohitukine; romurtide;roquinimex; rubiginone B 1; ruboxyl; safingol; saintopin; SarCNU;sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescencederived inhibitor 1; sense oligonucleotides; signal transductioninhibitors; signal transduction modulators; single-chain antigen bindingprotein; sizofiran; sobuzoxane; sodium borocaptate; sodiumphenylacetate; solverol; somatomedin binding protein; sonermin;sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin1; squalamine; stem cell inhibitor; stem-cell division inhibitors;stipiamide; stromelysin inhibitors; sulfinosine; superactive vasoactiveintestinal peptide antagonist; suradista; suramin; swainsonine;synthetic glycosaminoglycans; tallimustine; tamoxifen methiodide;tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium;telomerase inhibitors; temoporfin; temozolomide; teniposide;tetrachlorodecaoxide; tetrazomine; thaliblastine; thalidomide;thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin;thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone;tin ethyl etiopurpurin; tirapazamine; titanocene dichloride; topotecan;topsentin; toremifene; totipotent stem cell factor; translationinhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate;triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors;tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growthinhibitory factor; urokinase receptor antagonists; vapreotide; variolinB; vector system, erythrocyte gene therapy; velaresol; veramine;verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole;zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer.

Labeled Anti-TNFR2 Polypeptides

In some embodiments, antagonistic TNFR2 single-chain polypeptides,antibodies, antigen-binding fragments thereof, or constructs describedherein (e.g., any one or more of antibodies 1-25 described in Table 1and variants thereof, such as an antibody or antigen-binding fragmentcontaining one or more, or all, of the CDRs set forth in Table 1) areconjugated to another molecule (e.g., an epitope tag) for the purpose ofpurification or detection. Examples of such molecules that are useful inprotein purification include those that present structural epitopescapable of being recognized by a second molecule. This is a commonstrategy that is employed in protein purification by affinitychromatography, in which a molecule is immobilized on a solid supportand exposed to a heterogeneous mixture containing a target proteinconjugated to a molecule capable of binding the immobilized compound.Examples of epitope tag molecules that can be conjugated to antagonisticTNFR2 polypeptides for the purposes of molecular recognition include,without limitation, maltose-binding protein, glutathione-S-transferase,a poly-histidine tag, a FLAG-tag, a myc-tag, human influenzahemagglutinin (HA) tag, biotin, streptavidin. Conjugates containing theepitopes presented by these molecules are capable of being recognized bysuch complementary molecules as maltose, glutathione, anickel-containing complex, an anti-FLAG antibody, an anti-myc antibody,an anti-HA antibody, streptavidin, or biotin, respectively. For example,one can purify an antagonistic TNFR2 antibody or fragment thereofdescribed herein that has been conjugated to an epitope tag from acomplex mixture of other proteins and biomolecules (e.g., DNA, RNA,carbohydrates, phospholipids, etc) by treating the mixture with a solidphase resin containing an complementary molecule that can selectivelyrecognize and bind the epitope tag of the antagonistic anti-TNFR2antibody or fragment thereof. Examples of solid phase resins includeagarose beads, which are compatible with purifications in aqueoussolution.

An antagonistic TNFR2 polypeptide described herein can also becovalently appended to a fluorescent molecule, e.g., to detect theantibody or antigen-binding fragment thereof by fluorimetry and/or bydirect visualization using fluorescence microscopy. Exemplaryfluorescent molecules that can be conjugated to antibodies describedherein include green fluorescent protein, cyan fluorescent protein,yellow fluorescent protein, red fluorescent protein, phycoerythrin,allophycocyanin, hoescht, 4′,6-diamidino-2-phenylindole (DAPI),propidium iodide, fluorescein, coumarin, rhodamine,tetramethylrhodamine, and cyanine. Additional examples of fluorescentmolecules suitable for conjugation to antibodies described herein arewell-known in the art and have been described in detail in, e.g., U.S.Pat. Nos. 7,417,131 and 7,413,874, each of which is incorporated byreference herein.

Antagonistic TNFR2 polypeptides containing a fluorescent molecule areparticularly useful for monitoring the cell-surface localizationproperties of antibodies and fragments thereof described herein. Forinstance, one can expose cultured mammalian cells (e.g., T-reg cells) toantagonistic TNFR2 polypeptides described herein that have beencovalently conjugated to a fluorescent molecule and subsequently analyzethese cells using conventional fluorescent microscopy techniques knownin the art. Confocal fluorescent microscopy is a particularly powerfulmethod for determining cell-surface localization of antagonistic TNFR2polypeptides, as individual planes of a cell can be analyzed in order todistinguish antibodies or fragments thereof that have been internalizedinto a cell's interior, e.g., by receptor-mediated endocytosis, fromthose that are bound to the external face of the cell membrane.Additionally, cells can be treated with antagonistic TNFR2 antibodiesconjugated to a fluorescent molecule that emits visible light of aparticular wavelength (e.g., fluorescein, which fluoresces at about 535nm) and an additional fluorescent molecule that is known to localize toa particular site on the T-reg cell surface and that fluoresces at adifferent wavelength (e.g., a molecule that localizes to CD25 and thatfluoresces at about 599 nm). The resulting emission patterns can bevisualized by confocal fluorescence microscopy and the images from thesetwo wavelengths can be merged in order to reveal information regardingthe location of the antagonistic TNFR2 antibody or antigen-bindingfragment thereof on the T-reg cell surface with respect to otherreceptors.

Bioluminescent proteins can also be incorporated into a fusion proteinfor the purposes of detection and visualization of an antagonistic TNFR2polypeptide, such as a single-chain polypeptide, antibody, or fragmentthereof. Bioluminescent proteins, such as Luciferase and aequorin, emitlight as part of a chemical reaction with a substrate (e.g., luciferinand coelenterazine). Exemplary bioluminescent proteins suitable for useas a diagnostic sequence and methods for their use are described in,e.g., U.S. Pat. Nos. 5,292,658, 5,670,356, 6,171,809, and 7,183,092,each of which is herein incorporated by reference. Antagonistic TNFR2antibodies or fragments thereof labeled with bioluminescent proteins area useful tool for the detection of antibodies described herein followingan in vitro assay. For instance, the presence of an antagonistic TNFR2antibody that has been conjugated to a bioluminescent protein can bedetected among a complex mixture of additional proteins by separatingthe components of the mixture using gel electrophoresis methods known inthe art (e.g., native gel analysis) and subsequently transferring theseparated proteins to a membrane in order to perform a Western blot.Detection of the antagonistic TNFR2 polypeptide among the mixture ofother proteins can be achieved by treating the membrane with anappropriate Luciferase substrate and subsequently visualizing themixture of proteins on film using established protocols.

The polypeptides (e.g., single-chain polypeptides, antibodies,antigen-binding fragments thereof, and constructs thereof) describedherein can also be conjugated to a molecule comprising a radioactivenucleus, such that an antibody or fragment thereof described herein canbe detected by analyzing the radioactive emission pattern of thenucleus. Alternatively, an antagonistic TNFR2 antibody or fragmentthereof can be modified directly by incorporating a radioactive nucleuswithin the antibody during the preparation of the protein. Radioactiveisotopes of methionine (³⁵S), nitrogen (¹⁵N), or carbon (¹³C) can beincorporated into antibodies or fragments thereof described herein by,e.g., culturing bacteria in media that has been supplemented withnutrients containing these isotopes. Optionally, tyrosine derivativescontaining a radioactive halogen can be incorporated into anantagonistic TNFR2 polypeptide by, e.g., culturing bacterial cells inmedia supplemented with radiolabeled tyrosine. It has been shown thattyrosine functionalized with a radioactive halogen at the C2 position ofthe phenol system are rapidly incorporated into elongating polypeptidechains using the endogenous translation enzymes in vivo (U.S. Pat. No.4,925,651; incorporated herein by reference). The halogens includefluorine, chlorine, bromine, iodine, and astatine. Additionally,antagonistic TNFR2 polypeptides can be modified following isolation andpurification from cell culture by functionalizing polypeptides describedherein with a radioactive isotope. The halogens represent a class ofisotopes that can be readily incorporated into a purified protein byaromatic substitution at tyrosine or tryptophan, e.g., via reaction ofone or more of these residues with an electrophilic halogen species.Examples of radioactive halogen isotopes include ¹⁸F, ⁷⁵Br, ⁷⁷Br, ¹²²I,¹²³I, ¹²⁴I, ¹²⁵I, ¹²⁹I, ¹³¹I, or ²¹¹At.

Another alternative strategy for the incorporation of a radioactiveisotope is the covalent attachment of a chelating group to theantagonistic TNFR2 polypeptide, such as a single-chain polypeptide,antibody, fragment thereof, or construct. Chelating groups can becovalently appended to an antagonistic TNFR2 antibody or fragmentthereof by attachment to a reactive functional group, such as a thiol,amino group, alcohol, or carboxylic acid. The chelating groups can thenbe modified to contain any of a variety of metallic radioisotopes,including, without limitation, such radioactive nuclides as ¹²⁵I, ⁶⁷Ga,¹¹¹In, ⁹⁹Tc, ¹⁶⁹Yb, ¹⁸⁶Re, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ^(99m)Tc, ¹¹¹In,⁶⁴Cu, ⁶⁷Cu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁷⁷Lu, ⁹⁰Y, ⁷⁷As, ⁷²As, ⁸⁶Y, ⁸⁹Zr, ²¹¹At,²¹²Bi, ²¹³Bi, or ²²⁵Ac.

In some embodiments, it may be desirable to covalently conjugate thepolypeptides (e.g., single-chain polypeptides, antibodies, fragmentsthereof, or construct thereof) described herein with a chelating groupcapable of binding a metal ion from heavy elements or rare earth ions,such as Gd³⁺, Fe³⁺, Mn³⁺, or Cr²⁺. Conjugates containing chelatinggroups that are coordinated to such paramagnetic metals are useful as inMRI imaging applications. Paramagnetic metals include, but are notlimited to, chromium (III), manganese (II), iron (II), iron (III),cobalt (II), nickel (II), copper (II), praseodymium (III), neodymium(III), samarium (III), gadolinium (III), terbium (III), dysprosium(III), holmium (III), erbium (III), and ytterbium (III). In this way,antagonistic TNFR2 polypeptides can be detected by MRI spectroscopy. Forinstance, one can administer antagonistic TNFR2 antibodies or fragmentsthereof conjugated to chelating groups bound to paramagnetic ions to amammalian subject (e.g., a human patient) in order to monitor thedistribution of the antibody following administration. This can beachieved by administration of the antibody to a patient by any of theadministration routes described herein, such as intravenously, andsubsequently analyzing the location of the administered antibody byrecording an MRI of the patient according to established protocols.

Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides,antibodies, antigen-binding fragments thereof, and constructs thereof)can additionally be conjugated to other molecules for the purpose ofimproving the solubility and stability of the protein in aqueoussolution. Examples of such molecules include PEG, PSA, bovine serumalbumin (BSA), and human serum albumin (HSA), among others. Forinstance, one can conjugate an antagonistic TNFR2 polypeptide tocarbohydrate moieties in order to evade detection of the antibody orfragment thereof by the immune system of the patient receivingtreatment. This process of hyperglycosylation reduces the immunogenicityof therapeutic proteins by sterically inhibiting the interaction of theprotein with B cell receptors in circulation. Alternatively,antagonistic TNFR2 antibodies or fragments thereof can be conjugated tomolecules that prevent clearance from human serum and improve thepharmacokinetic profile of antibodies described herein. Exemplarymolecules that can be conjugated to or inserted within anti-TNFR2antibodies or fragments thereof described herein so as to attenuateclearance and improve the pharmacokinetic profile of these antibodiesand fragments include salvage receptor binding epitopes. These epitopesare found within the Fc region of an IgG immunoglobulin and have beenshown to bind Fc receptors and prolong antibody half-life in humanserum. The insertion of salvage receptor binding epitopes intoanti-TNFR2 antibodies or fragments thereof can be achieved, e.g., asdescribed in U.S. Pat. No. 5,739,277; incorporated herein by reference.

Modified Antagonistic TNFR2 Polypeptides

In addition to conjugation to other therapeutic agents and labels foridentification or visualization, antagonistic TNFR2 polypeptides (e.g.,single-chain polypeptides, antibodies, antigen-binding fragmentsthereof, and constructs thereof) described herein (e.g., any one or moreof antibodies 1-25 described in Table 1 and variants thereof, such as anantibody or antigen-binding fragment containing one or more, or all, ofthe CDRs set forth in Table 1) can also be modified so as to improvetheir pharmacokinetic profile, biophysical stability, or inhibitorycapacity. For instance, any cysteine residue not involved in maintainingthe proper conformation of the antagonistic TNFR2 polypeptide may besubstituted with an isosteric or isoelectronic amino acid (e.g., serine)in order to improve the oxidative stability of the molecule and preventaberrant crosslinking. Conversely, cystine bond(s) may be added to theantibody or fragment thereof to improve its stability (particularlywhere the antibody is an antibody fragment, such as an Fv fragment).This can be accomplished, e.g., by altering a polynucleotide encodingthe antibody heavy and light chains or a polynucleotide encoding anantibody fragment so as to encode one or more additional pairs ofcysteine residues that can form disulfide bonds under oxidativeconditions in order to reinforce antibody tertiary structure (see, e.g.,U.S. Pat. No. 7,422,899; incorporated herein by reference).

Another useful modification that may be made to antagonistic TNFR2polypeptides (e.g., single-chain polypeptides, antibodies,antigen-binding fragments thereof, and constructs thereof) describedherein includes altering the glycosylation profile of these antibodiesand fragments thereof. This can be achieved, e.g., by substituting,inserting, or deleting amino acids in an antagonistic TNFR2 antibody soas to insert or remove a glycosylation site. Glycosylation of antibodiestypically occurs in N-linked or O-linked fashion. N-linked glycosylationis a process whereby the attachment of a carbohydrate moiety to anantibody occurs at the side-chain of an asparagine residue. Consensusamino acid sequences for N-linked glycosylation include the tripeptidesequences asparagine-X-serine (NXS) and asparagine-X-threonine (NXT),where X is any amino acid except proline. The insertion of either ofthese tripeptide sequences in a polypeptide (e.g., an antagonistic TNFR2antibody) creates a potential glycosylation site. O-linked glycosylationrefers to the attachment of one of the sugars N-acetylgalactosamine,galactose, or xylose to a hydroxyamino acid, most commonly serine orthreonine, although 5-hydroxyproline or 5-hydroxylysine are alsocompetent substrates for glycoside formation. Addition of glycosylationsites to an anti-TNFR2 antibody can thus be accomplished by altering theamino acid sequence of the antibody (e.g., using recombinant expressiontechniques as described herein) such that it contains one or more of theabove-described tripeptide sequences to promote N-linked glycosylation,or one or more serine or threonine residues to the sequence of theoriginal antibody engender O-linked glycosylation (see, e.g., U.S. Pat.No. 7,422,899; incorporated herein by reference).

In alternative cases, it may be desirable to modify the antibody orfragment thereof described herein with respect to effector function,e.g., so as to enhance antigen-dependent cell-mediated cytotoxicity(ADCC) and/or complement dependent cytotoxicity (CDC) of the antibody.This may be achieved by introducing one or more amino acid substitutionsin an Fc region of the antibody. For instance, cysteine residues may beintroduced in the Fc region of an anti-TNFR2 antibody or fragmentthereof (e.g., by recombinant expression techniques as describedherein), so as to facilitate additional inter-chain disulfide bondformation in this region. The homodimeric antibody thus generated mayhave increased conformational constraint, which may foster improvedinternalization capability and/or increased complement-mediated cellkilling and antibody-dependent cellular cytotoxicity (ADCC). Homodimericantibodies with enhanced anti-tumor activity may also be prepared usingheterobifunctional cross-linkers as described, for example, in Wolff etal. (Canc. Res., 53:2560-2565, 1993); incorporated herein by reference.Alternatively, an antibody can be engineered which has dual Fc regionsand may thereby have enhanced complement lysis and ADCC capabilities(see Stevenson et al. (Anti-Canc. Drug Des., 3:219-230, 1989);incorporated herein by reference).

The serum half-life of antagonistic TNFR2 polypeptides (e.g.,single-chain polypeptides, antibodies, and antigen-binding fragmentsthereof) described herein can be improved in some embodiments byincorporating one more amino acid modifications, such as by altering theCH1 or CL region of the Fab domain to introduce a salvage receptormotif, e.g., that found in the two loops of a CH2 domain of an Fc regionof an IgG. Such alterations are described, for instance, in U.S. Pat.Nos. 5,869,046 and 6,121,022; incorporated herein by reference.Additional framework modifications can also be made to reduceimmunogenicity of the antibody or fragment thereof or to reduce orremove T cell epitopes that reside therein, as described for instance inUS2003/0153043; incorporated herein by reference.

Methods of Treatment

Antagonistic TNFR2 polypeptides, such a dominant antagonistic TNFR2polypeptide described herein (e.g., any one or more of antibodies 1-25described in Table 1 and variants thereof, such as an antibody orantigen-binding fragment containing one or more, or all, of the CDRs setforth in Table 1), can be used to treat a patient suffering from a cellproliferation disorder (such as a cancer described herein), aninfectious disease (such as a viral, bacterial, fungal, or parasiticinfection described herein), or another disease mediated by TNFR2signaling. These indications are explained in detail in the sectionsthat follow.

Methods of Treating Cell Proliferation Disorders

Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides,antibodies, antigen-binding fragments thereof, and constructs thereof)described herein, such as dominant antagonistic TNFR2 polypeptides, areuseful therapeutics for the treatment of a wide array of cancers andcell proliferation disorders. Antagonistic TNFR2 polypeptides (e.g.,single-chain polypeptides, antibodies, antigen-binding fragmentsthereof, and constructs thereof), such as dominant antagonistic TNFR2polypeptides, can be administered to a mammalian subject, such as ahuman, suffering from a cell proliferation disorder, such as cancer,e.g., to enhance the effectiveness of the adaptive immune responseagainst the target cancer cells.

Exemplary compositions of the disclosure that can be used for thesepurposes include antagonistic TNFR2 polypeptides (e.g., any one or moreof antibodies 1-25 described in Table 1 and variants thereof, such as anantibody or antigen-binding fragment containing one or more, or all, ofthe CDRs set forth in Table 1), such as those with at least two TNFR2binding sites in which the binding sites are spatially separated fromone another by about 133 Å or more, as well as those having a human IgG2isotype, for example, a human IgG2-A isotype (e.g., antagonistic TNFR2antibodies, antigen-binding fragments thereof, and constructs thereofhaving a human IgG2 hinge region having a C232S and/or C233S amino acidsubstitution). Compositions of the disclosure that can be used for thesepurposes also include pharmaceutical compositions containingantagonistic TNFR2 polypeptides that adopt a single disulfide-bondedisoform, such as those in which, e.g., 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, or more, of thepolypeptide in the pharmaceutical composition is present in a singledisulfide-bonded isoform.

In particular, antagonistic TNFR2 polypeptides (e.g., single-chainpolypeptides, antibodies, antigen-binding fragments thereof, andconstructs thereof) described herein (e.g., any one or more ofantibodies 1-25 described in Table 1 and variants thereof, such as anantibody or antigen-binding fragment containing one or more, or all, ofthe CDRs set forth in Table 1) can be administered to a mammaliansubject, such as a human, to inhibit T-reg cell growth and activation,which allows tumor-infiltrating T lymphocytes to localize to cellspresenting tumor-associated antigens and to promote cytotoxicity. Inaddition, polypeptides described herein may synergize with existingadoptive T cell therapy platforms, as one of the limitations on theeffectiveness of this strategy has been the difficulty of prolongingcytotoxicity of tumor-reactive T cells following infusion into amammalian subject (e.g., a human). Polypeptides described herein mayalso promote the activity of allogeneic T lymphocytes, which may expressforeign MHC proteins and may be increasingly susceptible to inactivationby the host immune system. For example, antagonistic TNFR2 polypeptidesdescribed herein can mitigate the T-reg-mediated depletion oftumor-reactive T cells by suppressing the growth and proliferation ofT-reg cells that typically accompanies T cell infusion. For instance,polypeptides (e.g., single-chain polypeptides, antibodies,antigen-binding fragments thereof, and constructs thereof) describedherein may be capable of reducing the growth of T-reg cells by about 50%to about 200% relative to untreated cells (e.g., 50%, 75%, 100%, 125%,150%, 175%, or 200%). The reduction in cellular growth does not requirethe presence of TNFα. In some embodiments, polypeptides (e.g.,single-chain polypeptides, antibodies, antigen-binding fragmentsthereof, and constructs thereof) described herein may be capable ofrestricting the growth of T-reg cells in the presence of TNFα to between90% and 150% relative to untreated cells (e.g., 90%, 100%, 110%, 120%,130%, 140%, or 150%). Antagonistic TNFR2 polypeptides (e.g.,single-chain polypeptides, antibodies, antigen-binding fragmentsthereof, and constructs thereof) described herein are also capable ofrestricting the proliferation of T-reg cells to less than 70% (e.g.,60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1%) of that of an untreatedpopulation of T-reg cells. Antagonistic TNFR2 polypeptides (e.g.,single-chain polypeptides, antibodies, antigen-binding fragmentsthereof, and constructs thereof) described herein are also capable ofdecreasing the survival of T-reg cells by about 10% (e.g., by about 20%,30%, 40%, or 50%, or more) relative to an untreated population of T-regcells.

Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides,antibodies, antigen-binding fragments thereof, and constructs thereof)described herein (e.g., any one or more of antibodies 1-25 described inTable 1 and variants thereof, such as an antibody or antigen-bindingfragment containing one or more, or all, of the CDRs set forth inTable 1) can be administered to a mammalian subject (e.g., a human)suffering from cancer in order to improve the condition of the patientby promoting the immune response against cancer cells and tumorogenicmaterial. Polypeptides described herein can be administered to asubject, e.g., via any of the routes of administration described herein.Polypeptides described herein can also be formulated with excipients,biologically acceptable carriers, and may be optionally conjugated to,admixed with, or co-administered separately (e.g., sequentially) withadditional therapeutic agents, such as anti-cancer agents. Cancers thatcan be treated by administration of antibodies or antigen-bindingfragments thereof described herein include such cancers as leukemia,lymphoma, liver cancer, bone cancer, lung cancer, brain cancer, bladdercancer, gastrointestinal cancer, breast cancer, cardiac cancer, cervicalcancer, uterine cancer, head and neck cancer, gallbladder cancer,laryngeal cancer, lip and oral cavity cancer, ocular cancer, melanoma,pancreatic cancer, prostate cancer, colorectal cancer, testicularcancer, and throat cancer. Particular cancers that can be treated byadministration of antibodies or antigen-binding fragments thereofdescribed herein include, without limitation, acute lymphoblasticleukemia (ALL), acute myeloid leukemia (AML), chronic lymphocyticleukemia (CLL), chronic myelogenous leukemia (CML), adrenocorticalcarcinoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer,appendix cancer, astrocytoma, atypical teratoid/rhabdoid tumor, basalcell carcinoma, bile duct cancer, extrahepatic cancer, ewing sarcomafamily, osteosarcoma and malignant fibrous histiocytoma, central nervoussystem embryonal tumors, central nervous system germ cell tumors,craniopharyngioma, ependymoma, bronchial tumors, burkitt lymphoma,carcinoid tumor, primary lymphoma, chordoma, chronic myeloproliferativeneoplasms, colon cancer, extrahepatic bile duct cancer, ductal carcinomain situ (DCIS), endometrial cancer, ependymoma, esophageal cancer,esthesioneuroblastoma, extracranial germ cell tumor, extragonadal germcell tumor, fallopian tube cancer, fibrous histiocytoma of bone,gastrointestinal carcinoid tumor, gastrointestinal stromal tumors(GIST), testicular germ cell tumor, gestational trophoblastic disease,glioma, childhood brain stem glioma, hairy cell leukemia, hepatocellularcancer, langerhans cell histiocytosis, hodgkin lymphoma, hypopharyngealcancer, islet cell tumors, pancreatic neuroendocrine tumors, wilms tumorand other childhood kidney tumors, langerhans cell histiocytosis, smallcell lung cancer, cutaneous T cell lymphoma, intraocular melanoma,merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer,midline tract carcinoma, multiple endocrine neoplasia syndromes,multiple myeloma/plasma cell neoplasm, myelodysplastic syndromes, nasalcavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma,non-hodgkin lymphoma (NHL), non-small cell lung cancer (NSCLC),epithelial ovarian cancer, germ cell ovarian cancer, low malignantpotential ovarian cancer, pancreatic neuroendocrine tumors,papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer,parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma,pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer,rectal cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer,kaposi sarcoma, rhabdomyosarcoma, sézary syndrome, small intestinecancer, soft tissue sarcoma, throat cancer, thymoma and thymiccarcinoma, thyroid cancer, transitional cell cancer of the renal pelvisand ureter, urethral cancer, endometrial uterine cancer, uterinesarcoma, vaginal cancer, vulvar cancer, and Waldenströmmacroglobulinemia.

For example, antagonistic TNFR2 polypeptides (e.g., single-chainpolypeptides, antibodies, antigen-binding fragments thereof, andconstructs thereof) described herein (e.g., any one or more ofantibodies 1-25 described in Table 1 and variants thereof, such as anantibody or antigen-binding fragment containing one or more, or all, ofthe CDRs set forth in Table 1) can be administered to a patient (e.g., amammalian patient, such as a human patient) in order to treat a cancercharacterized by TNFR2+ cancer cells, such as Hodgkin's lymphoma,cutaneous non-Hodgkin's lymphoma, T cell lymphoma, ovarian cancer, coloncancer, multiple myeloma, renal cell carcinoma, skin cancer, lungcancer, liver cancer, endometrial cancer, a hematopoietic or lymphoidcancer, a central nervous system cancer (e.g., glioma, blastoma, oranother cancer of the central nervous system described herein or knownin the art), breast cancer, pancreatic cancer, stomach cancer,esophageal cancer, and upper gastrointestinal cancer.

An antagonistic TNFR2 polypeptide described herein (e.g., any one ormore of antibodies 1-25 described in Table 1 and variants thereof, suchas an antibody or antigen-binding fragment containing one or more, orall, of the CDRs set forth in Table 1) can also be co-administered witha therapeutic antibody that exhibits reactivity towards a cancer cell.In this way, antagonistic TNFR2 polypeptides (e.g., single-chainpolypeptides, antibodies, antigen-binding fragments thereof, andconstructs thereof) described herein may synergize not only with theadaptive immune response, e.g., by prolonging T lymphocyte tumorreactivity, but also with other inhibitors of tumor cell growth.Examples of additional therapeutic antibodies that can be used to treatcancer and other cell proliferation disorders include those that exhibitreactivity with a tumor antigen or a cell-surface protein that isoverexpressed on the surface of a cancer cell. Exemplary antibodies thatcan be admixed, co-administered, or sequentially administered withantagonistic TNFR2 polypeptides described herein include, withoutlimitation, Trastuzumab (HERCEPTIN®), Bevacizumab (AVASTIN®), Cetuximab(ERBITUX®), Panitumumab (VECTIBIX®), Ipilimumab (YERVOY®), Rituximab(RITUXAN® and MABTHERA®), Alemtuzumab (CAMPATH®), Ofatumumab (ARZERRA®),Gemtuzumab ozogamicin (MYLOTARG®), Brentuximab vedotin (ADCETRIS®),⁹⁰Y-Ibritumomab Tiuxetan (ZEVALIN®), and ¹³¹I-Tositumomab (BEXXAR®),which are described in detail in Scott et al. (Cancer Immun., 12:14-21,2012); incorporated herein by reference.

A physician having ordinary skill in the art can readily determine aneffective amount of an antagonistic TNFR2 polypeptide, such assingle-chain polypeptide, antibody, antibody fragment, or constructdescribed herein (e.g., any one or more of antibodies 1-25 described inTable 1 and variants thereof, such as an antibody or antigen-bindingfragment containing one or more, or all, of the CDRs set forth in Table1), for administration to a mammalian subject (e.g., a human) in needthereof. For example, a physician could start prescribing doses of apolypeptide described herein at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved. Alternatively, a physicianmay begin a treatment regimen by administering an antagonistic TFNR2polypeptide, such as a single-chain polypeptide, antibody, antibodyfragment, or construct at a high dose and subsequently administerprogressively lower doses until a therapeutic effect is achieved (e.g.,a reduction in the volume of one or more tumors, a decrease in thepopulation of T-reg cells, or remission of a cell proliferationdisorder). In general, a suitable daily dose of a single-chainpolypeptide, antibody, antigen-binding fragment thereof, or constructdescribed herein will be an amount of the compound which is the lowestdose effective to produce a therapeutic effect. An antagonistic TNFR2polypeptide described herein may be administered, e.g., by injection,such as by intravenous, intramuscular, intraperitoneal, or subcutaneousinjection, optionally proximal to the site of the target tissue (e.g., atumor). A daily dose of a therapeutic composition of an antagonisticTNFR2 polypeptide described herein may be administered as a single doseor as two, three, four, five, six or more doses administered separatelyat appropriate intervals throughout the day, week, month, or year,optionally, in unit dosage forms. While it is possible for anantagonistic TNFR2 polypeptide described herein to be administeredalone, it may also be administered as a pharmaceutical formulation incombination with excipients, carriers, and optionally, additionaltherapeutic agents.

Polypeptides (e.g., single-chain polypeptides, antibodies,antigen-binding fragments thereof, and constructs thereof) describedherein (e.g., any one or more of antibodies 1-25 described in Table 1and variants thereof, such as an antibody or antigen-binding fragmentcontaining one or more, or all, of the CDRs set forth in Table 1) can bemonitored for their ability to attenuate the progression of a cellproliferation disease, such as cancer, by any of a variety of methodsknown in the art. For instance, a physician may monitor the response ofa mammalian subject (e.g., a human) to treatment with a polypeptide,such as a single-chain polypeptide, antibody, antibody fragment, orconstruct described herein by analyzing the volume of one or more tumorsin the patient. For example, polypeptides (e.g., single-chainpolypeptides, antibodies, antigen-binding fragments thereof, andconstructs thereof) described herein may be capable of reducing tumorvolume by between 1% and 100% (e.g., 1%, 5%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 99%, or 100%). Alternatively, a physician maymonitor the responsiveness of a subject (e.g., a human) to treatmentwith antagonistic TNFR2 polypeptides, such as single-chain polypeptides,antibodies, antigen-binding fragments thereof, or constructs describedherein by analyzing the T-reg cell population in the lymph of aparticular subject. For instance, a physician may withdraw a sample ofblood from a mammalian subject (e.g., a human) and determine thequantity or density of T-reg cells (e.g., CD4+ CD25+ FOXP3+T-reg cellsor CD17+ T-reg cells) using established procedures, such as fluorescenceactivated cell sorting.

Methods of Treating Infectious Diseases

Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides,antibodies, antigen-binding fragments thereof, and constructs thereof)described herein (e.g., any one or more of antibodies 1-25 described inTable 1 and variants thereof, such as an antibody or antigen-bindingfragment containing one or more, or all, of the CDRs set forth inTable 1) can also be used for treating infectious diseases, such asthose caused by any one or more of a virus, a bacterium, a fungus, or aparasite (e.g., a eukaryotic parasite). For instance, antagonistic TNFR2polypeptides (e.g., single-chain polypeptides, antibodies,antigen-binding fragments thereof, and constructs thereof) can beadministered to a mammalian subject (e.g., a human) suffering from aninfectious disease in order to treat the disease, as well as toalleviate one or more symptoms of the disease.

Exemplary compositions of the disclosure that can be used for thesepurposes include antagonistic TNFR2 polypeptides (e.g., any one or moreof antibodies 1-25 described in Table 1 and variants thereof, such as anantibody or antigen-binding fragment containing one or more, or all, ofthe CDRs set forth in Table 1), such as those with at least two TNFR2binding sites in which the binding sites are spatially separated fromone another by about 133 Å or more, as well as those having a human IgG2isotype, for example, a human IgG2-A isotype (e.g., antagonistic TNFR2antibodies, antigen-binding fragments thereof, and constructs thereofhaving a human IgG2 hinge region having a C232S and/or C233S amino acidsubstitution). Compositions of the disclosure that can be used for thesepurposes also include pharmaceutical compositions containingantagonistic TNFR2 polypeptides that adopt a single disulfide-bondedisoform, such as those in which, e.g., 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, or more, of thepolypeptide in the pharmaceutical composition is present in a singledisulfide-bonded isoform.

For example, antagonistic TNFR2 polypeptides (e.g., single-chainpolypeptides, antibodies, antigen-binding fragments thereof, andconstructs thereof) described herein (e.g., any one or more ofantibodies 1-25 described in Table 1 and variants thereof, such as anantibody or antigen-binding fragment containing one or more, or all, ofthe CDRs set forth in Table 1) can be used for treating, or alleviatingone or more symptoms of, viral infections in a mammalian subject, suchas a human, that are caused by, e.g., a member of the Flaviviridaefamily (e.g., a member of the Flavivirus, Pestivirus, and Hepacivirusgenera), which includes the hepatitis C virus, Yellow fever virus;Tick-borne viruses, such as the Gadgets Gully virus, Kadam virus,Kyasanur Forest disease virus, Langat virus, Omsk hemorrhagic fevervirus, Powassan virus, Royal Farm virus, Karshi virus, tick-borneencephalitis virus, Neudoerfl virus, Sofjin virus, Louping ill virus andthe Negishi virus; seabird tick-borne viruses, such as the Meaban virus,Saumarez Reef virus, and the Tyuleniy virus; mosquito-borne viruses,such as the Aroa virus, dengue virus, Kedougou virus, Cacipacore virus,Koutango virus, Japanese encephalitis virus, Murray Valley encephalitisvirus, St. Louis encephalitis virus, Usutu virus, West Nile virus,Yaounde virus, Kokobera virus, Bagaza virus, Ilheus virus, Israel turkeymeningoencephalo-myelitis virus, Ntaya virus, Tembusu virus, Zika virus,Banzi virus, Bouboui virus, Edge Hill virus, Jugra virus, Saboya virus,Sepik virus, Uganda S virus, Wesselsbron virus, yellow fever virus; andviruses with no known arthropod vector, such as the Entebbe bat virus,Yokose virus, Apoi virus, Cowbone Ridge virus, Jutiapa virus, Modocvirus, Sal Vieja virus, San Perlita virus, Bukalasa bat virus, CareyIsland virus, Dakar bat virus, Montana myotis leukoencephalitis virus,Phnom Penh bat virus, Rio Bravo virus, Tamana bat virus, and the Cellfusing agent virus; a member of the Arenaviridae family, which includesthe Ippy virus, Lassa virus (e.g., the Josiah, LP, or GA391 strain),lymphocytic choriomeningitis virus (LCMV), Mobala virus, Mopeia virus,Amapari virus, Flexal virus, Guanarito virus, Junin virus, Latino virus,Machupo virus, Oliveros virus, Paraná virus, Pichinde virus, Piritalvirus, Sabiá virus, Tacaribe virus, Tamiami virus, Whitewater Arroyovirus, Chapare virus, and Lujo virus; a member of the Bunyaviridaefamily (e.g., a member of the Hantavirus, Nairovirus, Orthobunyavirus,and Phlebovirus genera), which includes the Hantaan virus, Sin Nombrevirus, Dugbe virus, Bunyamwera virus, Rift Valley fever virus, La Crossevirus, California encephalitis virus, and Crimean-Congo hemorrhagicfever (CCHF) virus; a member of the Filoviridae family, which includesthe Ebola virus (e.g., the Zaire, Sudan, Ivory Coast, Reston, and Ugandastrains) and the Marburg virus (e.g., the Angola, Ci67, Musoke, Popp,Ravn and Lake Victoria strains); a member of the Togaviridae family(e.g., a member of the Alphavirus genus), which includes the Venezuelanequine encephalitis virus (VEE), Eastern equine encephalitis virus(EEE), Western equine encephalitis virus (WEE), Sindbis virus, rubellavirus, Semliki Forest virus, Ross River virus, Barmah Forest virus,O'nyong'nyong virus, and the chikungunya virus; a member of thePoxviridae family (e.g., a member of the Orthopoxvirus genus), whichincludes the smallpox virus, monkeypox virus, and vaccinia virus; amember of the Herpesviridae family, which includes the herpes simplexvirus (HSV; types 1, 2, and 6), human herpes virus (e.g., types 7 and8), cytomegalovirus (CMV), Epstein-Barr virus (EBV), Varicella-Zostervirus, and Kaposi's sarcoma associated-herpesvirus (KSHV); a member ofthe Orthomyxoviridae family, which includes the influenza virus (A, B,and C), such as the H5N1 avian influenza virus or H1N1 swine flu; amember of the Coronaviridae family, which includes the severe acuterespiratory syndrome (SARS) virus; a member of the Rhabdoviridae family,which includes the rabies virus and vesicular stomatitis virus (VSV); amember of the Paramyxoviridae family, which includes the humanrespiratory syncytial virus (RSV), Newcastle disease virus, hendravirus,nipahvirus, measles virus, rinderpest virus, canine distemper virus,Sendai virus, human parainfluenza virus (e.g., 1, 2, 3, and 4),rhinovirus, and mumps virus; a member of the Picornaviridae family,which includes the poliovirus, human enterovirus (A, B, C, and D),hepatitis A virus, and the coxsackievirus; a member of theHepadnaviridae family, which includes the hepatitis B virus; a member ofthe Papillamoviridae family, which includes the human papilloma virus; amember of the Parvoviridae family, which includes the adeno-associatedvirus; a member of the Astroviridae family, which includes theastrovirus; a member of the Polyomaviridae family, which includes the JCvirus, BK virus, and SV40 virus; a member of the Calciviridae family,which includes the Norwalk virus; a member of the Reoviridae family,which includes the rotavirus; and a member of the Retroviridae family,which includes the human immunodeficiency virus (HIV; e.g., types 1 and2), and human T lymphotropic virus Types I and II (HTLV-1 and HTLV-2,respectively); Friend Leukemia Virus; and transmissible spongiformencephalopathy, such as chronic wasting disease. Particularly, methodsdescribed herein include administering an antagonistic TNFR2 polypeptidedescribed herein to a human in order to treat an HIV infection (such asa human suffering from AIDS).

Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides,antibodies, antigen-binding fragments thereof, and constructs thereof)described herein (e.g., any one or more of antibodies 1-25 described inTable 1 and variants thereof, such as an antibody or antigen-bindingfragment containing one or more, or all, of the CDRs set forth inTable 1) can also be used for treating, or alleviating one or moresymptoms of, bacterial infections in a mammalian subject (e.g., ahuman). Examples of bacterial infections that may be treated byadministration of an antagonistic TNFR2 polypeptide, such as asingle-chain polypeptide, antibody, or antibody fragment describedherein include, without limitation, those caused by bacteria within thegenera Streptococcus, Bacillus, Listeria, Corynebacterium, Nocardia,Neisseria, Actinobacter, Moraxella, Enterobacteriacece (e.g., E. coli,such as O157:H7), Pseudomonas (such as Pseudomonas aeruginosa),Escherichia, Klebsiella, Serratia, Enterobacter, Proteus, Salmonella,Shigella, Yersinia, Haemophilus, Bordetella (such as Bordetellapertussis), Legionella, Pasteurella, Francisella, Brucella, Bartonella,Clostridium, Vibrio, Campylobacter, Staphylococcus, Mycobacterium (suchas Mycobacterium tuberculosis and Mycobacterium avium paratuberculosis,and Helicobacter (such as Helicobacter pylori and Helicobacterhepaticus). Particularly, methods described herein include administeringan antagonistic TNFR2 polypeptide, such as a single-chain polypeptide,antibody, antigen-binding fragment thereof, or construct that containsone or more, or all, of the CDR sequences of TNFRAB1, TNFRAB2, TNFRAB3,TNFRAB4, or TNFRAB5, such as a human, humanized, or chimeric variant ofTNFRAB1, TNFRAB2, TNFRAB3, TNFRAB4, or TNFRAB5, to a human or anon-human mammal in order to treat a Mycobacterium tuberculosisinfection. Particular methods described herein include administering anantagonistic TNFR2 polypeptide described herein to bovine mammals orbison in order to treat a Mycobacterium tuberculosis infection.Additionally, methods described herein include administering anantagonistic TNFR2 polypeptide described herein to a human or anon-human mammal in order to treat a Mycobacterium aviumparatuberculosis infection. Particular methods described herein includeadministering an antagonistic TNFR2 polypeptide described herein tobovine mammals or bison in order to treat a Mycobacterium aviumparatuberculosis infection.

Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides,antibodies, antigen-binding fragments thereof, and constructs thereof)described herein (e.g., any one or more of antibodies 1-25 described inTable 1 and variants thereof, such as an antibody or antigen-bindingfragment containing one or more, or all, of the CDRs set forth inTable 1) can also be administered to a mammalian subject (e.g., a human)for treating, or alleviating one or more symptoms of, parasiticinfections caused by a protozoan parasite (e.g., an intestinal protozoa,a tissue protozoa, or a blood protozoa) or a helminthic parasite (e.g.,a nematode, a helminth, an adenophorea, a secementea, a trematode, afluke (blood flukes, liver flukes, intestinal flukes, and lung flukes),or a cestode). Exemplary protozoan parasites that can be treatedaccording to the methods described herein include, without limitation,Entamoeba hystolytica, Giardia lamblia, Cryptosporidium muris,Trypanosomatida gambiense, Trypanosomatida rhodesiense, Trypanosomatidacrusi, Leishmania mexicana, Leishmania braziliensis, Leishmania tropica,Leishmania donovani, Leishmania major, Toxoplasma gondii, Plasmodiumvivax, Plasmodium ovale, Plasmodium malariae, Plasmodium falciparum,Plasmodium yoelli, Trichomonas vaginalis, and Histomonas meleagridis.Exemplary helminthic parasites include Richuris trichiura, Ascarislumbricoides, Enterobius vermicularis, Ancylostoma duodenale, Necatoramericanus, Strongyloides stercoralis, Wuchereria bancrofti, andDracunculus medinensis, Schistosoma mansoni, Schistosoma haematobium,Schistosoma japonicum, Fasciola hepatica, Fasciola gigantica,Heterophyes, Paragonimus westermani, Taenia solium, Taenia saginata,Hymenolepis nana, and Echinococcus granulosus. Additional parasiticinfections that can be treated according to the methods described hereininclude Onchocercas volvulus.

Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides,antibodies, and antigen-binding fragments thereof, such as any one ormore of antibodies 1-25 described in Table 1 and variants thereof, suchas an antibody or antigen-binding fragment containing one or more, orall, of the CDRs set forth in Table 1) can also be administered to amammalian subject (e.g., a human) in order to treat, or to alleviate oneor more symptoms of, fungal infections. Examples of fungal infectionsthat may be treated according to the methods described herein include,without limitation, those caused by, e.g., Aspergillus, Candida,Malassezia, Trichosporon, Fusarium, Acremonium, Rhizopus, Mucor,Pneumocystis, and Absidia. Exemplary fungal infections that can betreated according to the methods described herein also includePneumocystis carinii, Paracoccidioides brasiliensis and Histoplasmacapsulatum.

Pharmaceutical Compositions

Pharmaceutical compositions containing an antagonistic TNFR2polypeptide, such as a single-chain polypeptide, antibody,antigen-binding fragment thereof, or construct described herein can(e.g., any one or more of antibodies 1-25 described in Table 1 andvariants thereof, such as an antibody or antigen-binding fragmentcontaining one or more, or all, of the CDRs set forth in Table 1) beprepared using methods known in the art. Exemplary antagonistic TNFR2polypeptides that can be incorporated into pharmaceutical compositionsof the disclosure include those with at least two TNFR2 binding sites inwhich the binding sites are spatially separated from one another byabout 133 Å or more, as well as those having a human IgG2 isotype, forexample, a human IgG2-A isotype (e.g., antagonistic TNFR2 antibodies,antigen-binding fragments thereof, and constructs thereof having a humanIgG2 hinge region having a C232S and/or C233S amino acid substitution).

Pharmaceutical compositions described herein may contain an antagonisticTNFR2 polypeptide described herein (e.g., any one or more of antibodies1-25 described in Table 1 and variants thereof, such as an antibody orantigen-binding fragment containing one or more, or all, of the CDRs setforth in Table 1) in combination with one or more pharmaceuticallyacceptable excipients. For instance, pharmaceutical compositionsdescribed herein can be prepared using, e.g., physiologically acceptablecarriers, excipients or stabilizers (Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980); incorporated herein by reference),and in a desired form, e.g., in the form of lyophilized formulations oraqueous solutions. The compositions can also be prepared so as tocontain the active agent (e.g., an antagonistic anti-TNFR2 antibody orfragment thereof) at a desired concentration. For example, apharmaceutical composition described herein may contain at least 10%(e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%,99.5%, 99.9%, or 100%) active agent by weight (w/w).

Additionally, an active agent (e.g., an antagonistic TNFR2 polypeptidedescribed herein, such as a dominant antagonistic TNFR2 polypeptidedescribed herein) that can be incorporated into a pharmaceuticalformulation can itself have a desired level of purity. For example, apolypeptide, such as a single-chain polypeptide, antibody, orantigen-binding fragment thereof described herein may be characterizedby a certain degree of purity after isolating the antibody from cellculture media or after chemical synthesis, e.g., of a single-chainantibody fragment (e.g., scFv) by established solid phase peptidesynthesis methods or native chemical ligation as described herein. Anantagonistic TNFR2 polypeptide described herein may be at least 10% pureprior to incorporating the antibody into a pharmaceutical composition(e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%,99.5%, 99.9%, 99.99%, or 100% pure).

Additionally, antagonistic TNFR2 polypeptides of the disclosure (e.g.,any one or more of antibodies 1-25 described in Table 1 and variantsthereof, such as an antibody or antigen-binding fragment containing oneor more, or all, of the CDRs set forth in Table 1) may be incorporatedinto a pharmaceutical composition such that many of the polypeptidespresent in the pharmaceutical composition adopt a singledisulfide-bonded isoform. For example, pharmaceutical compositions ofthe disclosure include those containing an antagonist TNFR2 polypeptidein which, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%,97%, 98%, 99%, 99.9%, 99.99%, or more, of the polypeptide in thepharmaceutical composition is present in a single disulfide-bondedisoform, such as the IgG2-A isoform described herein.

Pharmaceutical compositions of antagonistic TNFR2 polypeptides describedherein (e.g., any one or more of antibodies 1-25 described in Table 1and variants thereof, such as an antibody or antigen-binding fragmentcontaining one or more, or all, of the CDRs set forth in Table 1) can beprepared for storage as lyophilized formulations or aqueous solutions bymixing the antibody having the desired degree of purity with optionalpharmaceutically acceptable carriers, excipients or stabilizerstypically employed in the art, e.g., buffering agents, stabilizingagents, preservatives, isotonifiers, non-ionic detergents, antioxidants,and other miscellaneous additives. See, e.g., Remington's PharmaceuticalSciences, 16th edition (Osol, ed. 1980; incorporated herein byreference). Such additives must be nontoxic to the recipients at thedosages and concentrations employed.

Buffering Agents

Buffering agents help to maintain the pH in the range which approximatesphysiological conditions. They can be present at concentration rangingfrom about 2 mM to about 50 mM. Suitable buffering agents for use withantagonistic TNFR2 polypeptides (e.g., single-chain polypeptides,antibodies, and antigen-binding fragments thereof) described hereininclude both organic and inorganic acids and salts thereof such ascitrate buffers {e.g., monosodium citrate-disodium citrate mixture,citric acid-trisodium citrate mixture, citric acid-monosodium citratemixture, etc.), succinate buffers {e.g., succinic acid-monosodiumsuccinate mixture, succinic acid-sodium hydroxide mixture, succinicacid-disodium succinate mixture, etc.), tartrate buffers (e.g., tartaricacid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture,tartaric acid-sodium hydroxide mixture, etc.), fumarate buffers {e.g.,fumaric acid-monosodium fumarate mixture, fumaric acid-disodium fumaratemixture, monosodium fumarate-disodium fumarate mixture, etc.), gluconatebuffers (e.g., gluconic acid-sodium gluconate mixture, gluconicacid-sodium hydroxide mixture, gluconic acid-potassium gluconatemixture, etc.), oxalate buffer (e.g., oxalic acid-sodium oxalatemixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassiumoxalate mixture, etc.), lactate buffers (e.g., lactic acid-sodiumlactate mixture, lactic acid-sodium hydroxide mixture, lacticacid-potassium lactate mixture, etc.) and acetate buffers {e.g., aceticacid-sodium acetate mixture, acetic acid-sodium hydroxide mixture,etc.). Additionally, phosphate buffers, histidine buffers andtrimethylamine salts such as Tris can be used.

Preservatives

Preservatives can be added to a composition described herein to retardmicrobial growth, and can be added in amounts ranging from 0.2%-1%(w/v). Suitable preservatives for use with antagonistic TNFR2polypeptides (e.g., single-chain polypeptides, antibodies,antigen-binding fragments thereof, and constructs thereof) describedherein include phenol, benzyl alcohol, meta-cresol, methyl paraben,propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkoniumhalides {e.g., chloride, bromide, and iodide), hexamethonium chloride,and alkyl parabens such as methyl or propyl paraben, catechol,resorcinol, cyclohexanol, and 3-pentanol. Isotonifiers sometimes knownas “stabilizers” can be added to ensure isotonicity of liquidcompositions described herein and include polyhydric sugar alcohols, forexample trihydric or higher sugar alcohols, such as glycerin, arabitol,xylitol, sorbitol and mannitol. Stabilizers refer to a broad category ofexcipients which can range in function from a bulking agent to anadditive which solubilizes the therapeutic agent or helps to preventdenaturation or adherence to the container wall. Typical stabilizers canbe polyhydric sugar alcohols (enumerated above); amino acids such asarginine, lysine, glycine, glutamine, asparagine, histidine, alanine,ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc.,organic sugars or sugar alcohols, such as lactose, trehalose, stachyose,mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glyceroland the like, including cyclitols such as inositol; polyethylene glycol;amino acid polymers; sulfur containing reducing agents, such as urea,glutathione, thioctic acid, sodium thioglycolate, thioglycerol,a-monothioglycerol and sodium thio sulfate; low molecular weightpolypeptides (e.g., peptides of 10 residues or fewer); proteins such ashuman serum albumin, bovine serum albumin, gelatin or immunoglobulins;hydrophilic polymers, such as polyvinylpyrrolidone monosaccharides, suchas xylose, mannose, fructose, glucose; disaccharides such as lactose,maltose, sucrose and trisaccharides such as raffinose; andpolysaccharides such as dextran. Stabilizers can be present in the rangefrom 0.1 to 10,000 weights per part of weight active protein.

Detergents

Non-ionic surfactants or detergents (also known as “wetting agents”) canbe added to help solubilize the therapeutic agent as well as to protectthe therapeutic protein against agitation-induced aggregation, whichalso permits the formulation to be exposed to shear surface stressedwithout causing denaturation of the protein. Suitable non-ionicsurfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188etc.), Pluronic polyols, polyoxyethylene sorbitan monoethers (TWEEN®-20,TWEEN®-80, etc.). Non-ionic surfactants can be present in a range ofabout 0.05 mg/mL to about 1.0 mg/mL, for example about 0.07 mg/mL toabout 0.2 mg/mL.

Additional miscellaneous excipients include bulking agents (e.g.,starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbicacid, methionine, vitamin E), and cosolvents.

Other Pharmaceutical Carriers

Alternative pharmaceutically acceptable carriers that can beincorporated into a pharmaceutical composition described herein mayinclude dextrose, sucrose, sorbitol, mannitol, starch, rubber arable,potassium phosphate, arginate, gelatin, potassium silicate,microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water,syrups, methyl cellulose, methyl hydroxy benzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils, but not limitedto. A composition containing an antagonistic TNFR2 antibody describedherein may further include a lubricant, a humectant, a sweetener, aflavoring agent, an emulsifier, a suspending agent, and a preservative.Details of suitable pharmaceutically acceptable carriers andformulations can be found in Remington's Pharmaceutical Sciences (19thed., 1995), which is incorporated herein by reference.

Compositions and Methods for Combination Therapy

Pharmaceutical compositions described herein may optionally include morethan one active agent. For instance, compositions described herein maycontain an antagonistic TNFR2 polypeptide (e.g., a single-chainpolypeptide, antibody, antigen-binding fragment thereof, or constructdescribed herein, such as any one or more of antibodies 1-25 describedin Table 1 and variants thereof, such as an antibody or antigen-bindingfragment containing one or more, or all, of the CDRs set forth inTable 1) conjugated to, admixed with, or administered separately fromanother pharmaceutically active molecule, e.g., a cytotoxic agent, anantibiotic, or a T lymphocyte (e.g., a gene-edited T lymphocyte for usein CAR-T therapy). For instance, an antagonistic TNFR2 polypeptide ortherapeutic conjugate thereof (e.g., a drug-antibody conjugate describedherein), may be admixed with one or more additional active agents thatcan be used to treat cancer or another cell proliferation disorder(e.g., neoplasm). Alternatively, pharmaceutical compositions describedherein may be formulated for co-administration or sequentialadministration with one or more additional active agents that can beused to treat cancer or other cell proliferation disorders. Examples ofadditional active agents that can be used to treat cancer and other cellproliferation disorders and that can be conjugated to, admixed with, oradministered separately from an antagonistic TNFR2 polypeptide describedherein include cytotoxic agents (e.g., those described herein), as wellas antibodies that exhibit reactivity with a tumor antigen or acell-surface protein that is overexpressed on the surface of a cancercell. Exemplary antibodies that can be conjugated to, admixed with, oradministered separately from antagonistic TNFR2 antibodies describedherein include, without limitation, Trastuzumab (HERCEPTIN®),Bevacizumab (AVASTIN®), Cetuximab (ERBITUX®), Panitumumab (VECTIBIX®),Ipilimumab (YERVOY®), Rituximab (RITUXAN® and MABTHERA®), Alemtuzumab(CAMPATH®), Ofatumumab (ARZERRA®), Gemtuzumab ozogamicin (MYLOTARG®),Brentuximab vedotin (ADCETRIS®), ⁹⁰Y-Ibritumomab Tiuxetan (ZEVALIN®),and ¹³¹I-Tositumomab (BEXXAR®), which are described in detail in Scottet al. (Cancer Immun., 12:14-21, 2012); incorporated herein byreference.

Additional agents that can be conjugated to, admixed with, oradministered separately from antagonistic TNFR2 polypeptides describedherein (e.g., any one or more of antibodies 1-25 described in Table 1and variants thereof, such as an antibody or antigen-binding fragmentcontaining one or more, or all, of the CDRs set forth in Table 1)include T lymphocytes that exhibit reactivity with a specific antigenassociated with a particular pathology. For instance, antagonistic TNFR2polypeptides described herein can be formulated for administration witha T cell that expresses a chimeric antigen receptor (CAR-T) in order totreat a cell proliferation disorder, such as a cancer described herein.Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides,antibodies, and antigen-binding fragments thereof) can synergize withCAR-T therapy by preventing T-reg cells from deactivating T lymphocytesthat have been genetically modified so as to express tumor-reactiveantigen receptors. In this way, CAR-T cells can be administered to apatient prior to, concurrently with, or after administration of anantagonistic TNFR2 polypeptide in order to treat a mammalian subject(e.g., a human) suffering from a cell proliferation disorder, such ascancer.

CAR-T therapy is a particularly robust platform for targeting cancercells in view of the ability to genetically engineer T lymphocytes toexpress an antigen receptor specific to a tumor-associated antigen. Forinstance, identification of antigens overexpressed on the surfaces oftumors and other cancer cells can inform the design and discovery ofchimeric T cell receptors, which are often composed of cytoplasmic andtransmembrane domains derived from a naturally-occurring T cell receptoroperatively linked to an extracellular scFv fragment that specificallybinds to a particular antigenic peptide. T cells can be geneticallymodified in order to express an antigen receptor that specifically bindsto a particular tumor antigen by any of a variety of genome editingtechniques described herein or known in the art. Exemplary techniquesfor modifying a T cell genome so as to incorporate a gene encoding achimeric antigen receptor include the CRISPER/Cas, zinc finger nuclease,TALEN, ARCUS™ platforms described herein. Methods for the geneticengineering of CAR-T lymphocytes have been described, e.g., in WO2014/127261, WO 2014/039523, WO 2014/099671, and WO 20120790000; thedisclosures of each of which are incorporated by reference herein.

CAR-T cells useful in the compositions and methods described hereininclude those that have been genetically modified such that the celldoes not express the endogenous T cell receptor. For instance, a CAR-Tcell may be modified by genome-editing techniques, such as thosedescribed herein, so as to suppress expression of the endogenous T cellreceptor in order to prevent graft-versus-host reactions in a patientreceiving a CAR-T infusion. Additionally, or alternatively, CAR-T cellscan be genetically modified so as to reduce the expression of one ormore endogenous MHC proteins. This is a particularly useful techniquefor the infusion of allogeneic T lymphocytes, as recognition of foreignMHC proteins represents one mechanism that promotes allograft rejection.One of skill in the art can also modify a T lymphocyte so as to suppressthe expression of immune suppressor proteins, such as programmed celldeath protein 1 (PD-1) and cytotoxic T lymphocyte-associated protein 4(CTLA-4). These proteins are cell surface receptors that, whenactivated, attenuate T cell activation. Infusion of CAR-T cells thathave been genetically modified so as to diminish the expression of oneor more immunosuppressor proteins represents one strategy that can beused to prolong the T lymphocyte-mediated cytotoxicity in vivo.

In addition to deleting specific genes, one can also modify CAR-T cellsin order to express a T cell receptor with a desired antigenspecificity. For instance, one can genetically modify a T lymphocyte inorder to express a T cell receptor that specifically binds to atumor-associated antigen in order to target infused T cells to cancercells. An exemplary T cell receptor that may be expressed by a CAR-Tcell is one that binds PD-L1, a cell surface protein that is oftenoverexpressed on various tumor cells. As PD-L1 activates PD-1 on thesurface of T lymphocytes, targeting this tumor antigen with CAR-Ttherapy can synergize with antagonistic TNFR2 antibodies or antibodyfragments described herein in order to increase the duration of animmune response mediated by a T lymphocyte in vivo. CAR-T cells can alsobe modified so as to express a T cell receptor that specifically bindsan antigen associated with one or more infectious disease, such as anantigen derived from a viral protein, a bacterial cell, a fungus, orother parasitic organism.

Other pharmaceutical compositions described herein include those thatcontain an antagonistic TNFR2 antibody or antibody fragment (e.g., anyone or more of antibodies 1-25 described in Table 1 and variantsthereof, such as an antibody or antigen-binding fragment containing oneor more, or all, of the CDRs set forth in Table 1), interferon alpha,and/or one or more antibiotics that can be administered to a patient(e.g., a human patient) suffering from an infectious disease. Forinstance, an antagonistic TNFR2 antibody or antibody fragment can beconjugated to, admixed with, or administered separately from anantibiotic useful for treating one or more infectious diseases, such asamikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin,paromomycin, streptomycin, spectinomycin, geldanamycin, herbimycin,rifaximin, loracarbef, ertapenem, doripenem, imipenem, meropenem,cefadroxil, cefazolin, cefazlexin, cefaclor, cefoxitin, cefprozil,cefuroxime, cefdinir, cefditoren, cefoperazone, clindamycin, lincomycin,daptomycin, erythromycin, linezolid, torezolid, amoxicillin, ampicillin,bacitracin, ciprofloxacin, doxycycline, and tetracycline, among others.

Immunotherapy Agents

An antagonistic TNFR2 polypeptide (e.g., a single-chain polypeptide,antibody, antigen-binding fragment thereof, or construct thereof)described herein (e.g., any one or more of antibodies 1-25 described inTable 1 and variants thereof, such as an antibody or antigen-bindingfragment containing one or more, or all, of the CDRs set forth inTable 1) may be admixed, conjugated, administered with, or administeredseparately from, an immunotherapy agent, for instance, for the treatmentof a cancer or infectious disease, such as a cancer or infectiousdisease described herein. Exemplary immunotherapy agents useful inconjunction with the compositions and methods described herein include,without limitation, an anti-CTLA-4 agent, an anti-PD-1 agent, ananti-PD-L1 agent, an anti-PD-L2 agent, an anti-TNF-α cross-linkingagent, an anti-TRAIL cross-linking agent, an anti-CD27 agent, ananti-CD30 agent, an anti-CD40 agent, an anti-4-1 BB agent, an anti-GITRagent, an anti-OX40 agent, an anti-TRAILR1 agent, an anti-TRAILR2 agent,and an anti-TWEAKR agent, as well as, for example, agents directedtoward the immunological targets described in Table 1 of Mahoney et al.,Cancer Immunotherapy, 14:561-584 (2015), the disclosure of which isincorporated herein by reference in its entirety. For example, theimmunotherapy agent may be an anti-CTLA-4 antibody or antigen-bindingfragment thereof, such as ipilimumab and tremelimumab. The immunotherapyagent may be an anti-PD-1 antibody or antigen-binding fragment thereof,such as nivolumab, pembrolizumab, avelumab, durvalumab, andatezolizumab. The immunotherapy agent may be an anti-PD-L1 antibody orantigen-binding fragment thereof, such as atezolizumab or avelumab. Asother examples, immunological target 4-1 BB ligand may be targeted withan anti-4-1 BB ligand antibody; immunological target OX40L may betargeted with an anti-OX40L antibody; immunological target GITR may betargeted with an anti-GITR antibody; immunological target CD27 may betargeted with an anti-CD27 antibody; immunological target TL1A may betargeted with an anti-TL1A antibody; immunological target CD40L or CD40may be targeted with an anti-CD40L antibody; immunological target LIGHTmay be targeted with an anti-LIGHT antibody; immunological target BTLAmay be targeted with an anti-BTLA antibody; immunological target LAG3may be targeted with an anti-LAG3 antibody; immunological target TIM3may be targeted with an anti-TIM3 antibody; immunological targetSinglecs may be targeted with an anti-Singlecs antibody; immunologicaltarget ICOS ligand may be targeted with an anti-ICOS ligand antibody;immunological target B7-H3 may be targeted with an anti-B7-H3 antibody;immunological target B7-H4 may be targeted with an anti-B7-H4 antibody;immunological target VISTA may be targeted with an anti-VISTA antibody;immunological target TMIGD2 may be targeted with an anti-TMIGD2antibody; immunological target BTNL2 may be targeted with an anti-BTNL2antibody; immunological target CD48 may be targeted with an anti-CD48antibody; immunological target KIR may be targeted with an anti-KIRantibody; immunological target LIR may be targeted with an anti-LIRantibody; immunological target ILT may be targeted with an anti-ILTantibody; immunological target NKG2D may be targeted with an anti-NKG2Dantibody; immunological target NKG2A may be targeted with an anti-NKG2Aantibody; immunological target MICA may be targeted with an anti-MICAantibody; immunological target MICB may be targeted with an anti-MICBantibody; immunological target CD244 may be targeted with an anti-CD244antibody; immunological target CSF1R may be targeted with an anti-CSF1Rantibody; immunological target IDO may be targeted with an anti-IDOantibody; immunological target TGFβ may be targeted with an anti-TGFβantibody; immunological target CD39 may be targeted with an anti-CD39antibody; immunological target CD73 may be targeted with an anti-CD73antibody; immunological target CXCR4 may be targeted with an anti-CXCR4antibody; immunological target CXCL12 may be targeted with ananti-CXCL12 antibody; immunological target SIRPA may be targeted with ananti-SIRPA antibody; immunological target CD47 may be targeted with ananti-CD47 antibody; immunological target VEGF may be targeted with ananti-VEGF antibody; and immunological target neuropilin may be targetedwith an anti-neuropilin antibody (see, e.g., Table 1 of Mahoney et al.).

Immunotherapy agents that may be used in conjunction with thecompositions and methods described herein include, for instance, ananti-TWEAK agent, an anti-cell surface lymphocyte protein agent, ananti-BRAF agent, an anti-MEK agent, an anti-CD33 agent, an anti-CD20agent, an anti-HLA-DR agent, an anti-HLA class I agent, an anti-CD52agent, an anti-A33 agent, an anti-GD3 agent, an anti-PSMA agent, ananti-Ceacan 1 agent, an anti-Galedin 9 agent, an anti-HVEM agent, ananti-VISTA agent, an anti-B7 H4 agent, an anti-HHLA2 agent, ananti-CD155 agent, an anti-CD80 agent, an anti-BTLA agent, an anti-CD160agent, an anti-CD28 agent, an anti-CD226 agent, an anti-CEACAM1 agent,an anti-TIM3 agent, an anti-TIGIT agent, an anti-CD96 agent, ananti-CD70 agent, an anti-CD27 agent, an anti-LIGHT agent, an anti-CD137agent, an anti-DR4 agent, an anti-CR5 agent, an anti-TNFRS agent, ananti-TNFR1 agent, an anti-FAS agent, an anti-CD95 agent, an anti-TRAILagent, an anti-DR6 agent, an anti-EDAR agent, an anti-NGFR agent, ananti-OPG agent, an anti-RANKL agent, an anti-LTβ receptor agent, ananti-BCMA agent, an anti-TACI agent, an anti-BAFFR agent, an anti-EDAR2agent, an anti-TROY agent, and an anti-RELT agent. For instance, theimmunotherapy agent may be an anti-TWEAK antibody or antigen-bindingfragment thereof, an anti-cell surface lymphocyte protein antibody orantigen-binding fragment thereof, an anti-BRAF antibody orantigen-binding fragment thereof, an anti-MEK antibody orantigen-binding fragment thereof, an anti-CD33 antibody orantigen-binding fragment thereof, an anti-CD20 antibody orantigen-binding fragment thereof, an anti-HLA-DR antibody orantigen-binding fragment thereof, an anti-HLA class I antibody orantigen-binding fragment thereof, an anti-CD52 antibody orantigen-binding fragment thereof, an anti-A33 antibody orantigen-binding fragment thereof, an anti-GD3 antibody orantigen-binding fragment thereof, an anti-PSMA antibody orantigen-binding fragment thereof, an anti-Ceacan 1 antibody orantigen-binding fragment thereof, an anti-Galedin 9 antibody orantigen-binding fragment thereof, an anti-HVEM antibody orantigen-binding fragment thereof, an anti-VISTA antibody orantigen-binding fragment thereof, an anti-B7 H4 antibody orantigen-binding fragment thereof, an anti-HHLA2 antibody orantigen-binding fragment thereof, an anti-CD155 antibody orantigen-binding fragment thereof, an anti-CD80 antibody orantigen-binding fragment thereof, an anti-BTLA antibody orantigen-binding fragment thereof, an anti-CD160 antibody orantigen-binding fragment thereof, an anti-CD28 antibody orantigen-binding fragment thereof, an anti-CD226 antibody orantigen-binding fragment thereof, an anti-CEACAM1 antibody orantigen-binding fragment thereof, an anti-TIM3 antibody orantigen-binding fragment thereof, an anti-TIGIT antibody orantigen-binding fragment thereof, an anti-CD96 antibody orantigen-binding fragment thereof, an anti-CD70 antibody orantigen-binding fragment thereof, an anti-CD27 antibody orantigen-binding fragment thereof, an anti-LIGHT antibody orantigen-binding fragment thereof, an anti-CD137 antibody orantigen-binding fragment thereof, an anti-DR4 antibody orantigen-binding fragment thereof, an anti-CR5 antibody orantigen-binding fragment thereof, an anti-TNFRS antibody orantigen-binding fragment thereof, an anti-TNFR1 antibody orantigen-binding fragment thereof, an anti-FAS antibody orantigen-binding fragment thereof, an anti-CD95 antibody orantigen-binding fragment thereof, an anti-TRAIL antibody orantigen-binding fragment thereof, an anti-DR6 antibody orantigen-binding fragment thereof, an anti-EDAR antibody orantigen-binding fragment thereof, an anti-NGFR antibody orantigen-binding fragment thereof, an anti-OPG antibody orantigen-binding fragment thereof, an anti-RANKL antibody orantigen-binding fragment thereof, an anti-LTβ receptor antibody orantigen-binding fragment thereof, an anti-BCMA antibody orantigen-binding fragment thereof, an anti-TACI antibody orantigen-binding fragment thereof, an anti-BAFFR antibody orantigen-binding fragment thereof, an anti-EDAR2 antibody orantigen-binding fragment thereof, an anti-TROY antibody orantigen-binding fragment thereof, or an anti-RELT antibody orantigen-binding fragment thereof.

In some embodiments, the immunotherapy agent is an anti-cell surfacelymphocyte protein antibody or antigen-binding fragment thereof, such asan antibody or antigen-binding fragment thereof that binds one or moreof CD1, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11, CD12, CD13,CD14, CD15, CD16, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25,CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37,CD38, CD39, CD40, CD41, CD42, CD43, CD44, CD45, CD46, CD47, CD48, CD49,CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60, CD61,CD62, CD63, CD64, CD65, CD66, CD67, CD68, CD69, CD70, CD71, CD72, CD73,CD74, CD75, CD76, CD77, CD78, CD79, CD80, CD81, CD82, CD83, CD84, CD85,CD86, CD87, CD88, CD89, CD90, CD91, CD92, CD93, CD94, CD95, CD96, CD97,CD98, CD99, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107,CD108, CD109, CD110, CD111, CD112, CD113, CD114, CD115, CD116, CD117,CD118, CD119, CD120, CD121, CD122, CD123, CD124, CD125, CD126, CD127,CD128, CD129, CD130, CD131, CD132, CD133, CD134, CD135, CD136, CD137,CD138, CD139, CD140, CD141, CD142, CD143, CD144, CD145, CD146, CD147,CD148, CD149, CD150, CD151, CD152, CD153, CD154, CD155, CD156, CD157,CD158, CD159, CD160, CD161, CD162, CD163, CD164, CD165, CD166, CD167,CD168, CD169, CD170, CD171, CD172, CD173, CD174, CD175, CD176, CD177,CD178, CD179, CD180, CD181, CD182, CD183, CD184, CD185, CD186, CD187,CD188, CD189, CD190, CD191, CD192, CD193, CD194, CD195, CD196, CD197,CD198, CD199, CD200, CD201, CD202, CD203, CD204, CD205, CD206, CD207,CD208, CD209, CD210, CD211, CD212, CD213, CD214, CD215, CD216, CD217,CD218, CD219, CD220, CD221, CD222, CD223, CD224, CD225, CD226, CD227,CD228, CD229, CD230, CD231, CD232, CD233, CD234, CD235, CD236, CD237,CD238, CD239, CD240, CD241, CD242, CD243, CD244, CD245, CD246, CD247,CD248, CD249, CD250, CD251, CD252, CD253, CD254, CD255, CD256, CD257,CD258, CD259, CD260, CD261, CD262, CD263, CD264, CD265, CD266, CD267,CD268, CD269, CD270, CD271, CD272, CD273, CD274, CD275, CD276, CD277,CD278, CD279, CD280, CD281, CD282, CD283, CD284, CD285, CD286, CD287,CD288, CD289, CD290, CD291, CD292, CD293, CD294, CD295, CD296, CD297,CD298, CD299, CD300, CD301, CD302, CD303, CD304, CD305, CD306, CD307,CD308, CD309, CD310, CD311, CD312, CD313, CD314, CD315, CD316, CD317,CD318, CD319, and/or CD320.

In some embodiments, the immunotherapy agent is an agent (e.g., apolypeptide, antibody, antigen-binding fragment thereof, a single-chainpolypeptide, or construct thereof) that binds a chemokine or lymphokine,such as a chemokine or lymphokine involved in tumor growth. Forinstance, exemplary immunotherapy agents that may be used in conjunctionwith the compositions and methods described herein include agents (e.g.,polypeptides, antibodies, antigen-binding fragments thereof,single-chain polypeptides, and constructs thereof) that bind and inhibitthe activity of one or more, or all, of CXCL1, CXCL2, CXCL3, CXCL8, CCL2and CCL5. Exemplary chemokines involved in tumor growth and that may betargeted using an immunotherapy agent as described herein include thosedescribed, for instance, in Chow et al., Cancer Immunol. Res.,2:1125-1131, 2014, the disclosure of which is incorporated herein byreference. Exemplary immunotherapy agents that may be used inconjunction with the compositions and methods described hereinadditionally include agents (e.g., polypeptides, antibodies,antigen-binding fragments thereof, single-chain polypeptides, andconstructs thereof) that bind and inhibit the activity of one or more,or all, of CCL3, CCL4, CCL8, and CCL22, which are described, forinstance, in Balkwill, Nat. Rev. Cancer, 4:540-550, 2004, the disclosureof which is incorporated herein by reference.

Additional examples of immunotherapy agents that can be used inconjunction with the compositions and methods described herein includeTargretin, Interferon-alpha, clobestasol, Peg Interferon (e.g.,PEGASYS®), prednisone, Romidepsin, Bexarotene, methotrexate,Trimcinolone cream, anti-chemokines, Vorinostat, gabapentin, antibodiesto lymphoid cell surface receptors and/or lymphokines, antibodies tosurface cancer proteins, and/or small molecular therapies likeVorinostat.

Using the methods described herein, an antagonistic TNFR2 polypeptide(e.g., a single-chain polypeptide, antibody, antigen-binding fragmentthereof, or construct thereof) described herein (e.g., any one or moreof antibodies 1-25 described in Table 1 and variants thereof, such as anantibody or antigen-binding fragment containing one or more, or all, ofthe CDRs set forth in Table 1) may be co-administered with (e.g.,admixed with) or administered separately from an immunotherapy agent.For example, an antagonistic TNFR2 polypeptide described herein (such asa single-chain polypeptide, antibody, antigen-binding fragment thereof,or construct described herein) may be administered to a patient, such asa human patient suffering from a cancer or infectious disease,simultaneously or at different times. In some embodiments, theantagonistic TNFR2 polypeptide (e.g., a single-chain polypeptide,antibody, antigen-binding fragment thereof, or construct describedherein) is administered to the patient prior to administration of animmunotherapy agent to the patient. Alternatively, the antagonisticTNFR2 polypeptide (e.g., a single-chain polypeptide, antibody,antigen-binding fragment thereof, or construct described herein) may beadministered to the patient after an immunotherapy agent. For example,the antagonistic TNFR2 polypeptide (e.g., a single-chain polypeptide,antibody, antigen-binding fragment thereof, or construct describedherein) may be administered to the patient after a failed immunotherapytreatment. A physician of skill in the art can monitor the efficacy ofimmunotherapy treatment to determine whether the therapy hassuccessfully ameliorated the pathology being treated (such as a canceror infectious disease, e.g., a cancer or infectious disease describedherein) using methods described herein and known in the art.

For instance, a physician of skill in the art may monitor the quantityof cancer cells in a sample isolated from a patient (e.g., a bloodsample or biopsy sample), such as a human patient, for instance, usingflow cytometry or FACS analysis. Additionally, or alternatively, aphysician of skill in the art can monitor the progression of a cancerousdisease in a patient, for instance, by monitoring the size of one ormore tumors in the patient, for example, by CT scan, MRI, or X-rayanalysis. A physician of skill in the art may monitor the progression ofa cancer, such as a cancer described herein, by evaluating the quantityand/or concentration of tumor biomarkers in the patient, such as thequantity and/or concentration of cell surface-bound tumor associatedantigens or secreted tumor antigens present in the blood of the patientas an indicator of tumor presence. A finding that the quantity of cancercells, the size of a tumor, and/or the quantity or concentration of oneor more tumor antigens present in the patient or in a sample isolatedfrom the patient has not decreased, for instance, by a statisticallysignificant amount following administration of the immunotherapy agentwithin a specified time period (e.g., from 1 day to 6 months, such as 1day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4weeks, 2 months, 3 months, 4 months, 5 months, or 6 months) can indicatethat the immunotherapy treatment has failed to ameliorate the cancer.Based on this indication, a physician of skill in the art may administeran antagonistic TNFR2 polypeptide described herein, such as asingle-chain polypeptide, antibody, antigen-binding fragment thereof, orconstruct described herein. Similarly, a physician a physician of skillin the art may monitor the quantity of bacterial, fungal, or parasiticcells, or the quantity of viral particles in a sample isolated from apatient suffering from an infectious disease, such as an infectiousdisease described herein. Additionally, or alternatively, a physician ofskill in the art may monitor the progression of an infectious disease byevaluating the symptoms of a patient suffering from such a pathology.For instance, a physician may monitor the patient by determining whetherthe frequency and/or severity of one or more symptoms of the infectiousdisease have stabilized (e.g., remained the same) or decreased followingtreatment with an immunotherapy agent. A finding that the quantity ofbacterial, fungal, or parasitic cells or viral particles in a sampleisolated from the patient and/or a finding that the frequency orseverity of one or more symptoms of the infectious disease have notdecreased, for instance, by a statistically significant amount followingadministration of the immunotherapy agent within a specified time period(e.g., from 1 day to 6 months, such as 1 day, 2 days, 3 days, 4 days, 5days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4months, 5 months, or 6 months) can indicate that the immunotherapytreatment has failed to ameliorate the infectious disease. Based on thisindication, a physician of skill in the art may administer anantagonistic TNFR2 polypeptide described herein, such as a single-chainpolypeptide, antibody, antigen-binding fragment thereof, or constructdescribed herein.

Chemotherapy Agents and Radiation Therapy

Additionally, or alternatively, an antagonistic TNFR2 polypeptide (e.g.,a single-chain polypeptide, antibody, antigen-binding fragment thereof,or construct thereof) described herein (e.g., any one or more ofantibodies 1-25 described in Table 1 and variants thereof, such as anantibody or antigen-binding fragment containing one or more, or all, ofthe CDRs set forth in Table 1) may be admixed, conjugated, administeredwith, or administered separately from, a chemotherapy agent, forexample, for the treatment of cancer, such as a cancer described herein.Exemplary chemotherapy agents useful in conjunction with thecompositions and methods described herein include, without limitation,Abiraterone Acetate, ABITREXATE® (Methotrexate), ABRAXANE® (PaclitaxelAlbumin), ADRIAMYCIN®, bleomycin, vinblastine, and dacarbazine (ABVD),ADRIAMYCIN®, bleomycin, vincristine sulfate, and etoposide phosphate(ABVE), ADRIAMYCIN®, bleomycin, vincristine sulfate, etoposidephosphate, prednisone, and cyclophosphamide (ABVE-PC), doxorubicin andcyclophosphamide (AC), doxorubicin, cyclophosphamide, and paclitaxel ordocetaxel (AC-T), ADCETRIS® (Brentuximab Vedotin), cytarabine,daunorubicin, and etoposide (ADE), ado-trastuzumab emtansine,ADRIAMYCIN® (doxorubicin hydrochloride), afatinib dimaleate, AFINITOR®(Everolimus), AKYNZEO® (netupitant and palonosetron hydrochloride),ALDARA® (imiquimod), aldesleukin, ALECENSA® (alectinib), alectinib,alemtuzumab, ALKERAN® for Injection (Melphalan Hydrochloride), ALKERAN®tablets (melphalan), ALIMTA® (pemetrexed disodium), ALOXI® (palonosetronhydrochloride), AMBOCHLORIN® (chlorambucil), AMBOCLORIN® (Chlorambucil),aminolevulinic acid, anastrozole, aprepitant, AREDIA® (pamidronatedisodium), ARIMIDEX® (anastrozole), AROMASIN® (exemestane), ARRANON®(nelarabine), arsenic trioxide, ARZERRA® (ofatumumab), asparaginaseErwinia chrysanthemi, AVASTIN® (bevacizumab), axitinib, azacitidine,BEACOPP Becenum (carmustine), BELEODAQ® (Belinostat), belinostat,bendamustine hydrochloride, bleomycin, etoposide, and cisplatin (BEP),bevacizumab, bexarotene, BEXXAR® (tositumomab and iodine ¹³¹Itositumomab), bicalutamide, BiCNU (carmustine), bleomycin, blinatumomab,BLINCYTO® (blinatumomab), bortezomib, BOSULIF® (bosutinib), bosutinib,brentuximab vedotin, busulfan, BUSULFEX® (busulfan), cabazitaxel,cabozantinib-S-malate, CAF, CAM PATH® (alemtuzumab), CAMPTOSAR®(irinotecan hydrochloride), capecitabine, CAPDX, CARAC® (fluorouracil),carboplatin, CARBOPLATIN-TAXOL®, carfilzomib, CARMUBRIS® (carmustine),carmustine, carmustine implant, CASODEX® (bicalutamide), CEENU(lomustine), cisplatin, etoposide, and methotrexate (CEM), ceritinib,CERUBIDINE® (daunorubicin hydrochloride), CERVARIX® (recombinant HPVbivalent vaccine), cetuximab, chlorambucil, chlorambucil-prednisone,CHOP, cisplatin, CLAFEN® (cyclophosphamide), clofarabine, CLOFAREX®(clofarabine), CLOLAR® (Clofarabine), CMF, cobimetinib, cometriq(cabozantinib-S-malate), COPDAC, COPP, COPP-ABV, COSMEGEN®(dactinomycin), COTELLIC® (cobimetinib), crizotinib, CVP,cyclophosphamide, CYFOS® (ifosfamide), CYRAMZA® (ramucirumab),cytarabine, cytarabine liposome, CYTOSAR-U® (cytarabine), CYTOXAN®(cyclophosphamide), dabrafenib, dacarbazine, DACOGEN® (decitabine),dactinomycin, daratumumab, DARZALEX® (daratumumab), dasatinib,daunorubicin hydrochloride, decitabine, degarelix, denileukin diftitox,denosumab, DEPOCYT® (cytarabine liposome), dexamethasone, dexrazoxanehydrochloride, dinutuximab, docetaxel, DOXIL® (doxorubicinhydrochloride), doxorubicin hydrochloride, DOX-SL® (doxorubicinhydrochloride), DTIC-DOME® (dacarbazine), EFUDEX (fluorouracil), ELITEK®(rasburicase), ELLENCE® (epirubicin hydrochloride), elotuzumab,ELOXATIN® (oxaliplatin), eltrombopag olamine, EMEND® (aprepitant),EMPLICITI® (elotuzumab), enzalutamide, epirubicin hydrochloride, EPOCH,ERBITUX® (cetuximab), eribulin mesylate, ERIVEDGE® (vismodegib),erlotinib hydrochloride, ERWINAZE® (asparaginase Erwinia chrysanthemi),ETOPOPHOS® (etoposide phosphate), etoposide, etoposide phosphate,EVACET® (doxorubicin hydrochloride liposome), everolimus, EVISTA®(raloxifene hydrochloride), EVOMELA® (melphalan hydrochloride),exemestane, 5-FU (5-fluorouracil), FARESTON® (toremifene), FARYDAK®(panobinostat), FASLODEX® (fulvestrant), FEC, FEMARA® (letrozole),filgrastim, FLUDARA® (fludarabine phosphate), fludarabine phosphate,FLUOROPLEX® (fluorouracil), fluorouracil injection, flutamide, FOLEX®(methotrexate), FOLEX® PFS (methotrexate), FOLFIRI, FOLFIRI-bevacizumab,FOLFIRI-cetuximab, FOLFIRINOX, FOLFOX, FOLOTYN® (pralatrexate), FU-LV,fulvestrant, GARDASIL® (recombinant HPV quadrivalent vaccine), GARDASIL9® (recombinant HPV nonavalent vaccine), GAZYVA® (obinutuzumab),gefitinib, gemcitabine hydrochloride, gemcitabine-cisplatin,gemcitabine-oxaliplatin, gemtuzumab ozogamicin, GEMZAR® (gemcitabinehydrochloride), GILOTRIF® (afatinib dimaleate), GLEEVEC® (imatinibmesylate), GLIADEL® (carmustine implant), GLIADEL® wafer (carmustineimplant), glucarpidase, goserelin acetate, HALAVEN® (eribulin mesylate),HERCEPTIN® (trastuzumab), HPV bivalent vaccine, HYCAMTIN® (topotecanhydrochloride), Hyper-CVAD, IBRANCE (palbociclib), IBRITUMOMAB®tiuxetan, ibrutinib, ICE, ICLUSIG® (ponatinib hydrochloride), IDAMYCIN®(idarubicin hydrochloride), idarubicin hydrochloride, idelalisib, IFEX®(ifosfamide), ifosfamide, ifosfamidum, IL-2 (aldesleukin), imatinibmesylate, IMBRUVICA® (ibrutinib), ilmiquimod, IMLYGIC® (talimogenelaherparepvec), INLYTA (axitinib), recombinant interferon alpha-2b,intron A, tositumomab, such as ¹³¹I tositumomab, ipilimumab, IRESSA®(gefitinib), irinotecan hydrochloride, ISTODAX® (romidepsin),ixabepilone, ixazomib citrate, IXEMPRA® (ixabepilone), JAKAFI®(ruxolitinib phosphate), JEVTANA® (cabazitaxel), KADCYLA®(ado-trastuzumab emtansine), KEOXIFENE® (raloxifene hydrochloride),KEPIVANCE® (palifermin), KEYTRUDA® (pembrolizumab), KYPROLIS®(carfilzomib), lanreotide acetate, lapatinib ditosylate, lenalidomide,lenvatinib mesylate, LENVIMA® (lenvatinib mesylate), letrozole,leucovorin calcium, leukeran (chlorambucil), leuprolide acetate, levulan(aminolevulinic acid), LINFOLIZIN® (chlorambucil), LIPODOX® (doxorubicinhydrochloride liposome), lomustine, LONSURF® (trifluridine and tipiracilhydrochloride), LUPRON® (leuprolide acetate), LYNPARZA® (olaparib),MARQIBO® (vincristine sulfate liposome), MATULANE® (procarbazinehydrochloride), mechlorethamine hydrochloride, megestrol acetate,MEKINIST® (trametinib), melphalan, melphalan hydrochloride,mercaptopurine, MESNEX® (mesna), METHAZOLASTONE® (temozolomide),methotrexate, methotrexate LPF, MEXATE® (methotrexate), MEXATE-AQ®(methotrexate), mitomycin C, mitoxantrone hydrochloride, MITOZYTREX®(mitomycin C), MOPP, MOZOBIL® (plerixafor), MUSTARGEN® (mechlorethaminehydrochloride), MUTAMYCIN® (mitomycin C), MYLERAN® (busulfan), MYLOSAR®(azacitidine), MYLOTARG® (gemtuzumab ozogamicin), nanoparticlepaclitaxel, NAVELBINE® (vinorelbine tartrate), NECITUMUMAB, nelarabine,NEOSAR® (cyclophosphamide), netupitant and palonosetron hydrochloride,NEUPOGEN® (filgrastim), NEXAVAR® (sorafenib tosylate), NILOTINIB,NINLARO® (ixazomib citrate), nivolumab, NOLVADEX® (tamoxifen citrate),NPLATE® (romiplostim), obinutuzumab, ODOMZO® (sonidegib), OEPA,ofatumumab, OFF, olaparib, omacetaxine mepesuccinate, ONCASPAR®(pegaspargase), ondansetron hydrochloride, ONIVYDE® (irinotecanhydrochloride liposome), ONTAK® (denileukin diftitox), OPDIVO®(nivolumab), OPPA, osimertinib, oxaliplatin, paclitaxel, paclitaxelalbumin-stabilized nanoparticle formulation, PAD, palbociclib,palifermin, palonosetron hydrochloride, palonosetron hydrochloride andnetupitant, pamidronate disodium, panitumumab, panobinostat, PARAPLAT®(carboplatin), PARPLATIN® (carboplatin), pazopanib hydrochloride, PCV,pegaspargase, peginterferon alpha-2b, PEG-INTRON® (peginterferonalpha-2b), pembrolizumab, pemetrexed disodium, PERJETA® (pertuzumab),pertuzumab, PLATINOL® (cisplatin), PLATINOL-AQ® (cisplatin), plerixafor,pomalidomide, POMALYST® (pomalidomide), ponatinib hydrochloride,PORTRAZZA® (necitumumab), pralatrexate, prednisone, procarbazinehydrochloride, PROLEUKIN® (aldesleukin), PROLIA® (denosumab), PROMACTA(eltrombopag olamine), PROVENGE® (sipuleucel-T), PURINETHOL®(mercaptopurine), PURIXAN® (mercaptopurine), ²²³Ra dichloride,raloxifene hydrochloride, ramucirumab, rasburicase, R-CHOP, R-CVP,recombinant human papillomavirus (HPV), recombinant interferon alpha-2b,regorafenib, R-EPOCH, REVLIMID® (lenalidomide), RHEUMATREX®(methotrexate), RITUXAN® (rituximab), rolapitant hydrochloride,romidepsin, romiplostim, rubidomycin (daunorubicin hydrochloride),ruxolitinib phosphate, SCLEROSOL® intrapleural aerosol (talc),siltuximab, sipuleucel-T, somatuline depot (lanreotide acetate),sonidegib, sorafenib tosylate, SPRYCEL® (dasatinib), STANFORD V, steriletalc powder (talc), STERITALC® (talc), STIVARGA® (regorafenib),sunitinib malate, SUTENT® (sunitinib malate), SYLATRON® (peginterferonalpha-2b), SYLVANT® (siltuximab), SYNOVIR® (thalidomide), SYNRIBO®(omacetaxine mepesuccinate), thioguanine, TAC, TAFINLAR® (dabrafenib),TAGRISSO® (osimertinib), talimogene laherparepvec, tamoxifen citrate,tarabine PFS (cytarabine), TARCEVA (erlotinib hydrochloride), TARGRETIN®(bexarotene), TASIGNA® (nilotinib), TAXOL® (paclitaxel), TAXOTERE®(docetaxel), TEMODAR® (temozolomide), temsirolimus, thalidomide,THALOMID® (thalidomide), thioguanine, thiotepa, TOLAK® (topicalfluorouracil), topotecan hydrochloride, toremifene, TORISEL®(temsirolimus), TOTECT® (dexrazoxane hydrochloride), TPF, trabectedin,trametinib, TREANDA® (bendamustine hydrochloride), trifluridine andtipiracil hydrochloride, TRISENOX® (arsenic trioxide), TYKERB®(lapatinib ditosylate), UNITUXIN® (dinutuximab), uridine triacetate,VAC, vandetanib, VAMP, VARUBI® (rolapitant hydrochloride), vectibix(panitumumab), VeIP, VELBAN® (vinblastine sulfate), VELCADE®(bortezomib), VELSAR (vinblastine sulfate), VEMURAFENIB, VIADUR(leuprolide acetate), VIDAZA (azacitidine), vinblastine sulfate,VINCASAR® PFS (vincristine sulfate), vincristine sulfate, vinorelbinetartrate, VIP, vismodegib, VISTOGARD® (uridine triacetate), VORAXAZE®(glucarpidase), vorinostat, VOTRIENT® (pazopanib hydrochloride),WELLCOVORIN® (leucovorin calcium), XALKORI® (crizotinib), XELODA®(capecitabine), XELIRI, XELOX, XGEVA® (denosumab), XOFIGO® (²²³Radichloride), XTANDI® (enzalutamide), YERVOY® (ipilimumab), YONDELIS®(trabectedin), ZALTRAP® (ziv-aflibercept), ZARXIO® (filgrastim),ZELBORAF® (vemurafenib), ZEVALIN® (ibritumomab tiuxetan), ZINECARD®(dexrazoxane hydrochloride), ziv-aflibercept, ZOFRAN® (ondansetronhydrochloride), ZOLADEX® (gGoserelin acetate), zoledronic acid, ZOLINZA®(vorinostat), ZOMETA® (zoledronic acid), ZYDELIG® (idelalisib), ZYKADIA®(ceritinib), and ZYTIGA (abiraterone acetate).

Using the methods described herein, an antagonistic TNFR2 polypeptide(e.g., a single-chain polypeptide, antibody, antigen-binding fragmentthereof, or construct thereof) described herein may be co-administeredwith (e.g., admixed with) or administered separately from a chemotherapyagent for the treatment of cancer. For example, an antagonistic TNFR2polypeptide described herein (such as a single-chain polypeptide,antibody, antigen-binding fragment thereof, or construct describedherein) may be administered to a patient, such as a human patientsuffering from a cancer, simultaneously or at different times. In someembodiments, the antagonistic TNFR2 polypeptide (e.g., a single-chainpolypeptide, antibody, antigen-binding fragment thereof, or constructdescribed herein) is administered to the patient prior to administrationof a chemotherapy agent to the patient. Alternatively, the antagonisticTNFR2 polypeptide (e.g., a single-chain polypeptide, antibody,antigen-binding fragment thereof, or construct described herein) may beadministered to the patient after a chemotherapy agent. For example, theantagonistic TNFR2 polypeptide (e.g., a single-chain polypeptide,antibody, antigen-binding fragment thereof, or construct describedherein) may be administered to the patient after a failed chemotherapytreatment. A physician of skill in the art can monitor the efficacy ofchemotherapy treatment to determine whether the therapy has successfullyameliorated the pathology being treated (such as a cancer describedherein) using methods described herein and known in the art.

For instance, a physician of skill in the art may monitor the quantityof cancer cells in a sample isolated from a patient (e.g., a bloodsample or biopsy sample), such as a human patient, for instance, usingflow cytometry or FACS analysis. Additionally, or alternatively, aphysician of skill in the art can monitor the progression of a cancerousdisease in a patient, for instance, by monitoring the size of one ormore tumors in the patient, for example, by CT scan, MRI, or X-rayanalysis. A physician of skill in the art may monitor the progression ofa cancer, such as a cancer described herein, by evaluating the quantityand/or concentration of tumor biomarkers in the patient, such as thequantity and/or concentration of cell surface-bound tumor associatedantigens or secreted tumor antigens present in the blood of the patientas an indicator of tumor presence. A finding that the quantity of cancercells, the size of a tumor, and/or the quantity or concentration of oneor more tumor antigens present in the patient or a sample isolated fromthe patient has not decreased, for instance, by a statisticallysignificant amount following administration of the chemotherapy agentwithin a specified time period (e.g., from 1 day to 6 months, such as 1day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4weeks, 2 months, 3 months, 4 months, 5 months, or 6 months) can indicatethat the chemotherapy treatment has failed to ameliorate the cancer.Based on this indication, a physician of skill in the art may administeran antagonistic TNFR2 polypeptide described herein, such as asingle-chain polypeptide, antibody, antigen-binding fragment thereof, orconstruct described herein.

Additionally, or alternatively, an antagonistic TNFR2 polypeptide (e.g.,a single-chain polypeptide, antibody, antigen-binding fragment thereof,or construct thereof) described herein may be administeredsimultaneously with, or administered separately from, radiation therapy.For instance, a physician of skill in the art may administer radiationtherapy to a patient, such as a human patient suffering from a cancerdescribed herein, by treating the patient with external and/or internalelectromagnetic radiation. The energy delivered by such radiation, whichis typically in the form of X-rays, gamma rays, and similar forms oflow-wavelength energy, can cause oxidative damage to the DNA of cancercells, thereby leading to cell death, for instance, by apoptosis.External radiation therapy can be administered, for instance, usingmachinery such as a radiation beam to expose the patient to a controlledpulse of electromagnetic radiation. Additionally, or alternatively, thepatient may be administered internal radiation, for instance, byadministering to the patient a therapeutic agent that contains aradioactive substituent, such as agents that contain ²²³Ra or ¹³¹I,which emit high-energy alpha and beta particles, respectively. Exemplarytherapeutic agents that may be conjugated to a radiolabel include, forexample, small molecule chemotherapeutics, antibodies, andantigen-binding fragments thereof, among others. For instance, anantagonistic TNFR2 polypeptide (e.g., a single-chain polypeptide,antibody, antigen-binding fragment thereof, or construct thereof)described herein may be conjugated to a radioactive substituent or amoiety that ligate such a substituent, for example, using bond-formingtechniques known in the art or described herein. Such conjugates can beadministered to the subject in order to deliver a therapeutic dosage ofradiation therapy and a TNFR2 antagonist described herein in asimultaneous administration (see, for example, “Antagonistic TNFR2polypeptide conjugates,” above).

In some embodiments, the antagonistic TNFR2 polypeptide (e.g., asingle-chain polypeptide, antibody, antigen-binding fragment thereof, orconstruct described herein) is administered to the patient after failedradiation treatment. A physician of skill in the art can monitor theefficacy of radiation treatment to determine whether the therapy hassuccessfully ameliorated the pathology being treated (such as a cancerdescribed herein) using, e.g., methods described herein. For instance, aphysician of skill in the art may monitor the quantity of cancer cellsin a sample isolated from a patient (e.g., a blood sample or biopsysample), such as a human patient, for instance, using flow cytometry orFACS analysis. Additionally, or alternatively, a physician of skill inthe art can monitor the progression of a cancerous disease in a patient,for instance, by monitoring the size of one or more tumors in thepatient, for example, by CT scan, MRI, or X-ray analysis. A physician ofskill in the art may monitor the progression of a cancer, such as acancer described herein, by evaluating the quantity and/or concentrationof tumor biomarkers in the patient, such as the quantity and/orconcentration of cell surface-bound tumor associated antigens orsecreted tumor antigens present in the blood of the patient as anindicator of tumor presence. A finding that the quantity of cancercells, the size of a tumor, and/or the quantity or concentration of oneor more tumor antigens present in the patient or a sample isolated fromthe patient has not decreased, for instance, by a statisticallysignificant amount following administration of the radiation therapywithin a specified time period (e.g., from 1 day to 6 months, such as 1day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4weeks, 2 months, 3 months, 4 months, 5 months, or 6 months) can indicatethat the radiation treatment has failed to ameliorate the cancer. Basedon this indication, a physician of skill in the art may administer anantagonistic TNFR2 polypeptide described herein, such as a single-chainpolypeptide, antibody, antigen-binding fragment thereof, or constructdescribed herein.

In some embodiments, a physician of skill in the art may administer to apatient suffering from cancer a chemotherapeutic agent, radiationtherapy, and a TNFR2 antagonist described herein (such as a single-chainpolypeptide, antibody, antigen-binding fragment thereof, or constructdescribed herein). The TNFR2 antagonist described herein,chemotherapeutic agent, and radiation therapy may be administered to thepatient simultaneously (for instance, in a single pharmaceuticalcomposition or as multiple compositions administered to the patient atthe same time) or at different times. In some embodiments, the TNFR2antagonist (such as an antibody, antigen-binding fragment thereof,single-chain polypeptide, or construct described herein) is administeredto the patient first, and the chemotherapeutic agent and radiationtherapy follow. Alternatively, the TNFR2 antagonist (such as asingle-chain polypeptide, antibody, antigen-binding fragment thereof, orconstruct described herein) may be administered to the patient followingchemotherapy and radiation treatment. For example, the antagonisticTNFR2 polypeptide (e.g., a single-chain polypeptide, antibody,antigen-binding fragment thereof, or construct described herein) may beadministered to the patient after failed chemotherapy and/or radiationtreatment.

A physician of skill in the art can monitor the efficacy of chemotherapyand radiation treatment to determine whether the therapy hassuccessfully ameliorated the pathology being treated (such as a cancerdescribed herein) using methods described herein, such as the methodsdescribed above. For instance, a physician of skill in the art maymonitor the quantity of cancer cells in a sample isolated from a patient(e.g., a blood sample or biopsy sample), such as a human patient, forinstance, using flow cytometry or FACS analysis. Additionally, oralternatively, a physician of skill in the art can monitor theprogression of a cancerous disease in a patient, for instance, bymonitoring the size of one or more tumors in the patient, for example,by CT scan, MRI, or X-ray analysis. A physician of skill in the art maymonitor the progression of a cancer, such as a cancer described herein,by evaluating the quantity and/or concentration of tumor biomarkers inthe patient, such as the quantity and/or concentration of cellsurface-bound tumor associated antigens or secreted tumor antigenspresent in the blood of the patient as an indicator of tumor presenceand even measure serum soluble TNFR2. One skilled in the art wouldexpect a decrease in the number of activated T-regs, and increase in thenumbers of T effectors and a decrease in the total number of cancercells. Because of the specificity of these TNFR2 antibodies for cancer,the clinical monitoring would be expected to be most dramatic in thetumor microenvironment. A finding that the quantity of cancer cells, thesize of a tumor, and/or the quantity or concentration of one or moretumor antigens present in the patient or a sample isolated from thepatient has not decreased, for instance, by a statistically significantamount following administration of the chemotherapy agent and radiationwithin a specified time period (e.g., from 1 day to 6 months, such as 1day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4weeks, 2 months, 3 months, 4 months, 5 months, or 6 months) can indicatethat the chemotherapy and radiation treatment has failed to amelioratethe cancer. Based on this indication, a physician of skill in the artmay administer an antagonistic TNFR2 polypeptide described herein, suchas a single-chain polypeptide, antibody, antigen-binding fragmentthereof, or construct described herein.

Blood-Brain Barrier Penetration

In certain embodiments, antagonistic TNFR2 polypeptides (e.g.,single-chain polypeptides, antibodies, antigen-binding fragmentsthereof, and constructs thereof) described herein can be formulated toensure proper distribution in vivo. For example, the blood-brain barrier(BBB) excludes many highly hydrophilic compounds. To ensure that thetherapeutic compositions described herein cross the BBB (if desired),they can be formulated, for example, in liposomes. Methods ofmanufacturing liposomes have been described, e.g., U.S. Pat. Nos.4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise one ormore moieties that are selectively transported into specific cells ororgans, thereby enhancing targeted drug delivery (see, e.g., V. V.Ranade (J. Clin. Pharmacol. 29:685, 1989)). Exemplary targeting moietiesinclude, e.g., folate or biotin (see, e.g., U.S. Pat. No. 5,416,016);mannosides (Umezawa et al. (Biochem. Biophys. Res. Commun. 153:1038,1988)); antibodies (P. G. Bloeman et al. (FEBS Lett. 357:140, 1995); M.Owais et al. (Antimicrob. Agents Chemother. 39:180, 1995)); surfactantprotein A receptor (Briscoe et al. (Am. J. Physiol. 1233:134, 1995));the disclosures of each of which are incorporated herein by reference.

Routes of Administration and Dosing

Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides,antibodies, antigen-binding fragments thereof, and constructs thereof)described herein (e.g., any one or more of antibodies 1-25 described inTable 1 and variants thereof, such as an antibody or antigen-bindingfragment containing one or more, or all, of the CDRs set forth inTable 1) can be administered to a mammalian subject (e.g., a human) by avariety of routes such as orally, transdermally, subcutaneously,intranasally, intravenously, intramuscularly, intraocularly,intratumorally, parenterally, topically, intrathecally andintracerebroventricularly. The most suitable route for administration inany given case will depend on the particular polypeptide administered,the patient, pharmaceutical formulation methods, administration methods(e.g., administration time and administration route), the patients age,body weight, sex, severity of the diseases being treated, the patient'sdiet, and the patient's excretion rate.

The effective dose of an antagonistic TNFR2 polypeptide described hereincan range, for instance, from about 0.0001 to about 100 mg/kg of bodyweight per single (e.g., bolus) administration, multiple administrationsor continuous administration (e.g., a continuous infusion), or toachieve a serum concentration of 0.0001-5000 μg/mL serum concentrationper single (e.g., bolus) administration, multiple administrations orcontinuous administration (e.g., continuous infusion), or any effectiverange or value therein depending on the condition being treated, theroute of administration and the age, weight, and condition of thesubject. In certain embodiments, e.g., for the treatment of cancer, eachdose can range from about 0.0001 mg to about 500 mg/kg of body weight.For instance, a pharmaceutical composition described herein may beadministered in a daily dose in the range of 0.001-100 mg/kg (bodyweight). The dose may be administered one or more times (e.g., 2-10times) per day, week, month, or year to a mammalian subject (e.g., ahuman) in need thereof.

Antagonistic TNFR2 polypeptides described herein (e.g., single-chainpolypeptides, antibodies, antigen-binding fragments thereof, andconstructs thereof) can be administered to a patient by way of acontinuous intravenous infusion or as a single bolus administration. Theantagonistic TNFR2 polypeptides described herein (e.g., e.g.,single-chain polypeptides, antibodies, antigen-binding fragmentsthereof, and constructs thereof) may be administered to a patient in anamount of, for example, from 0.01 μg to about 5 g in a volume of, forexample, from 10 μL to 10 mL. The antagonistic TNFR2 polypeptides may beadministered to a patient over the course of several minutes to severalhours. For example, the antagonistic TNFR2 polypeptides described hereinmay be administered to a patient over the course of from 5 minutes to 5hours, such as over the course of 5 minutes, 10 minutes, 15 minutes, 20minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, 80 minutes, 90minutes, 95 minutes, 100 minutes, 105 minutes, 110 minutes, 115 minutes,120 minutes, 125 minutes, 130 minutes, 135 minutes, 140 minutes, 145minutes, 150 minutes, 155 minutes, 160 minutes, 165 minutes, 170minutes, 175 minutes, 180 minutes, 185 minutes, 190 minutes, 195minutes, 200 minutes, 205 minutes, 210 minutes, 215 minutes, 220minutes, 225 minutes, 230 minutes, 235 minutes, 240 minutes, 245minutes, 250 minutes, 255 minutes, 260 minutes, 265 minutes, 270minutes, 275 minutes, 280 minutes, 285 minutes, 290 minutes, 295minutes, or 300 minutes, or more.

Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides,antibodies, antigen-binding fragments thereof, and constructs thereof)described herein may be administered in combination with animmunotherapy agent, such as an anti-PD-1 antibody or antigen-bindingfragment thereof, an anti-PD-L1 antibody or antigen-binding fragmentthereof, and/or an anti-CTLA-4 antibody or antigen-binding fragmentthereof. Exemplary anti-PD-1 antibodies include nivolumab,pembrolizumab, avelumab, durvalumab, and atezolizumab. Exemplaryanti-CTLA4 antibodies include ipilimumab and tremelimumab. Exemplaryanti-PD-L1 antibodies include atezolizumab and avelumab.

When an anti-PD-1 antibody or antigen-binding fragment thereof isadministered to a patient (e.g., a patient having cancer or aninfectious disease described herein) in combination with an antagonistTNFR2 polypeptide, the anti-PD-1 antibody may be administered to thepatient by way of a single bolus administration or continuousintravenous infusion. For example, pembrolizumab may be administered toa human patient by way of a continuous intravenous infusion of 200 mgover the course of 30 minutes, for instance, every three weeks, asneeded (KEYTRUDA® (pembrolizumab) [package insert]. Merck Sharp & DohmeCorp., Whitehouse Station, NJ, the disclosure of which is incorporatedherein by reference in its entirety). In another example, nivolumab maybe administered to a patient by way of a continuous intravenous infusionof 240 mg over the course of 30 minutes, for instance, every two weeksas needed. Alternatively, nivolumab may be administered to a patient byway of a continuous intravenous infusion of 480 mg over the course of 30minutes, for instance, every four weeks as needed (OPDIVO® (nivolumab)[package insert]. Bristol-Myers Squibb Company, Princeton, N.J., thedisclosure of which is incorporated herein by reference in itsentirety).

When an anti-CTLA-4 antibody or antigen-binding fragment thereof isadministered to a patient (e.g., a patient having cancer or aninfectious disease described herein) in combination with an antagonistTNFR2 polypeptide, the anti-CTLA-4 antibody may be administered to thepatient by way of a single bolus administration or continuousintravenous infusion. For example, ipilimumab may be administered to ahuman patient by way of a continuous intravenous infusion of 3 mg/kgover the course of 90 minutes, for instance, every three weeks, asneeded, or by way of a continuous intravenous infusion of 10 mg/kg overthe course of 90 minutes every three weeks for four doses, followed by10 mg/kg over the course of 90 minutes every 12 weeks for up to 3 years(YERVOY® (ipilimumab) [package insert]. Bristol-Myers Squibb Company,Princeton, N.J., the disclosure of which is incorporated herein byreference in its entirety).

When TNFR2 antagonist polypeptides (e.g., single-chain polypeptides,antibodies, antigen-binding fragments thereof, and constructs thereof)are administered to a patient in combination with an immunotherapyagent, such as an anti-PD-1 antibody or anti-CTLA-4 antibody, theantagonist TNFR2 polypeptide and the immunotherapy agent may beco-administered to the patient, for example, by way of a continuousintravenous infusion or bolus administration of the first agent,followed by a continuous intravenous infusion or bolus administration ofthe second agent. The administration of the two agents may occurconcurrently. Alternatively, the administration of the antagonist TNFR2antibody or antigen-binding fragment thereof may precede or follow theadministration of the immunotherapy agent. In some embodiments,administration of the second agent (e.g., the antagonist TNFR2polypeptide) commences within from about 5 minutes to about 4 weeks, ormore, of the end of the administration of the first agent (e.g., theimmunotherapy agent). For example, administration of the second agentmay commence within about 5 minutes, 10 minutes, 20 minutes, 30 minutes,40 minutes, 50 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours,6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20hours, 21 hours, 22 hours, 23 hours, 24 hours, 2 days, 3 days, 4 days, 5days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, or more, of the end ofthe administration of the first agent.

Therapeutic compositions can be administered with medical devices knownin the art. For example, in an embodiment, a therapeutic compositiondescribed herein can be administered with a needleless hypodermicinjection device, such as the devices disclosed in U.S. Pat. Nos.5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or4,596,556. Examples of well-known implants and modules useful inconjunction with the compositions and methods described herein include:U.S. Pat. No. 4,487,603, which discloses an implantable micro-infusionpump for dispensing medication at a controlled rate; U.S. Pat. No.4,486,194, which discloses a therapeutic device for administeringmedicaments through the skin; U.S. Pat. No. 4,447,233, which discloses amedication infusion pump for delivering medication at a precise infusionrate; U.S. Pat. No. 4,447,224, which discloses a variable flowimplantable infusion apparatus for continuous drug delivery; U.S. Pat.No. 4,439,196, which discloses an osmotic drug delivery system havingmulti-chamber compartments; and U.S. Pat. No. 4,475,196, which disclosesan osmotic drug delivery system. These patents are incorporated hereinby reference. Many other such implants, delivery systems, and modulesare known to those skilled in the art.

Kits Containing Antagonistic Anti-TNFR2 Polypeptides

Also included herein are kits that contain antagonistic TNFR2polypeptides (e.g., single-chain polypeptides, antibodies,antigen-binding fragments thereof, and constructs thereof, such as anyone or more of antibodies 1-25 described in Table 1 and variantsthereof, such as an antibody or antigen-binding fragment containing oneor more, or all, of the CDRs set forth in Table 1). The kits providedherein may contain any of the antagonistic TNFR2 polypeptides describedabove, as well as any of the polynucleotides encoding thesepolypeptides, vectors containing these polynucleotides, or cellsengineered to express and secrete antibodies described herein (e.g.,prokaryotic or eukaryotic cells).

Exemplary compositions of the disclosure that can be incorporated into akit described herein include antagonistic TNFR2 polypeptides, such asthose with at least two TNFR2 binding sites in which the binding sitesare spatially separated from one another by about 133 Å or more, as wellas those having a human IgG2 isotype, for example, a human IgG2-Aisotype (e.g., antagonistic TNFR2 antibodies, antigen-binding fragmentsthereof, and constructs thereof having a human IgG2 hinge region havinga C232S and/or C233S amino acid substitution). Compositions of thedisclosure that can be incorporated into kits described herein alsoinclude pharmaceutical compositions containing antagonistic TNFR2polypeptides that adopt a single disulfide-bonded isoform, such as thosein which, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%,97%, 98%, 99%, 99.9%, 99.99%, or more, of the polypeptide in thepharmaceutical composition is present in a single disulfide-bondedisoform.

A kit described herein may include reagents that can be used to producethe compositions described herein (e.g., antagonistic TNFR2polypeptides, such as single-chain polypeptides, antibodies, constructs,conjugates containing antagonistic TNFR2 polypeptides, polynucleotidesencoding antagonistic anti-TNFR2 polypeptides, vectors containing thesepolynucleotides). Optionally, kits described herein may include reagentsthat can induce the expression of antagonistic TNFR2 polypeptides withincells (e.g., mammalian cells), such as doxycycline or tetracycline. Inother cases, a kit described herein may contain a compound capable ofbinding and detecting a fusion protein that contains an antagonisticTNFR2 antibody and an epitope tag. For instance, in such cases a kitdescribed herein may contain maltose, glutathione, a nickel-containingcomplex, an anti-FLAG antibody, an anti-myc antibody, an anti-HAantibody, biotin, or streptavidin.

Kits described herein may also include reagents that are capable ofdetecting an antagonistic TNFR2 polypeptide (e.g., single-chainpolypeptide, antibody, fragment thereof, or construct thereof) directly.Examples of such reagents include secondary antibodies that selectivelyrecognize and bind particular structural features within the Fc regionof an anti-TNFR2 antibody described herein. Kits described herein maycontain secondary antibodies that recognize the Fc region of anantagonistic TNFR2 antibody and that are conjugated to a fluorescentmolecule. These antibody-fluorophore conjugates provide a tool foranalyzing the localization of antagonistic anti-TNFR2 antibodies, e.g.,in a particular tissue or cultured mammalian cell using establishedimmunofluorescence techniques. In some embodiments, kits describedherein may include additional fluorescent compounds that exhibit knownsub-cellular localization patterns. These reagents can be used incombination with another antibody-fluorophore conjugate, e.g., one thatspecifically recognizes a different receptor on the cell surface inorder to analyze the localization of an anti-TNFR2 antibody relative toother cell-surface proteins.

Kits described herein may also contain a reagent that can be used forthe analysis of a patient's response to treatment by administration ofantagonistic TNFR2 polypeptides (e.g., single-chain polypeptides,antibodies, antigen-binding fragments thereof, and constructs thereof)described herein. For instance, kits described herein may include anantagonistic TNFR2 antibody and one or more reagents that can be used todetermine the quantity of T-reg cells in a blood sample withdrawn from asubject (e.g., a human) that is undergoing treatment with an antibodydescribed herein. Such a kit may contain, e.g., antibodies thatselectively bind cell-surface antigens presented by T-reg cells, such asCD4 and CD25. Optionally, these antibodies may be labeled with afluorescent dye, such as fluorescein or tetramethylrhodamine, in orderto facilitate analysis of T-reg cells by fluorescence-activated cellsorting (FACS) methods known in the art. Kits described herein mayoptionally contain one or more reagents that can be used to quantifytumor-reactive T lymphocytes in order to determine the effectiveness ofan antagonistic TNFR2 polypeptide described herein in restoringtumor-infiltrating lymphocyte proliferation. For instance, kitsdescribed herein may contain an antibody that selectively bindscell-surface markers on the surface of a cytotoxic T cell, such as CD8or CD3. Optionally, these antibodies may be labeled with fluorescentmolecules so as to enable quantitation by FACS analysis.

A kit described herein may also contain one or more reagents useful fordetermining the affinity and selectivity of an antagonistic TNFR2polypeptide described herein for one or more peptides derived from TNFR2(e.g., a peptide containing the sequence of any one of SEQ ID NOs: 11,19, 20, and 34-117). For instance, a kit may contain an antagonisticTNFR2 polypeptide and one or more reagents that can be used in an ELISAassay to determine the K_(D) of an antibody described herein for one ormore peptides that present a TNFR2 epitope in a conformation similar tothat of the epitope in the native protein. A kit may contain, e.g., amicrotiter plate containing wells that have been previously conjugatedto avidin, and may contain a library of TNFR2-derived peptides, each ofwhich conjugated to a biotin moiety. Such a kit may optionally contain asecondary antibody that specifically binds to the Fc region of anantagonistic TNFR2 antibody described herein, and the secondary antibodymay be conjugated to an enzyme (e.g., horseradish peroxidase) thatcatalyzes a chemical reaction that results in the emission ofluminescent light.

Kits described herein may also contain antagonistic TNFR2 polypeptidesdescribed herein and reagents that can be conjugated to such anantibody, including those previously described (e.g., a cytotoxic agent,a fluorescent molecule, a bioluminescent molecule, a molecule containinga radioactive isotope, a molecule containing a chelating group bound toa paramagnetic ion, etc). These kits may additionally containinstructions for how the conjugation of an antagonistic TNFR2 antibodydescribed herein to a second molecule, such as those described above,can be achieved.

A kit described herein may also contain a vector containing apolynucleotide that encodes an antagonistic TNFR2 polypeptide, such asany of the vectors described herein. Alternatively, a kit may includemammalian cells (e.g., CHO cells) that have been genetically altered toexpress and secrete antagonistic TNFR2 antibodies or fragments thereoffrom the nuclear genome of the cell. Such a kit may also containinstructions describing how expression of the antagonistic TNFR2antibody or fragment thereof from a polynucleotide can be induced, andmay additionally include reagents (such as, e.g., doxycycline ortetracycline) that can be used to promote the transcription of thesepolynucleotides. Such kits may be useful for the manufacture ofantagonistic TNFR2 antibodies or antigen-binding fragments thereofdescribed herein.

Other kits described herein may include tools for engineering aprokaryotic or eukaryotic cell (e.g., a CHO cell or a BL21(DE3) E. colicell) so as to express and secrete an antagonistic TNFR2 polypeptidedescribed herein from the nuclear genome of the cell. For example, a kitmay contain CHO cells stored in an appropriate media and optionallyfrozen according to methods known in the art. The kit may also provide avector containing a polynucleotide that encodes a nuclease (e.g., suchas the CRISPER/Cas, zinc finger nuclease, TALEN, ARCUS™ nucleasesdescribed herein) as well as reagents for expressing the nuclease in thecell. The kit can additionally provide tools for modifying thepolynucleotide that encodes the nuclease so as to enable one to alterthe DNA sequence of the nuclease in order to direct the cleavage of aspecific target DNA sequence of interest. Examples of such tools includeprimers for the amplification and site-directed mutagenesis of thepolynucleotide encoding the nuclease of interest. The kit may alsoinclude restriction enzymes that can be used to selectively excise thenuclease-encoding polynucleotide from the vector and subsequentlyre-introduce the modified polynucleotide back into the vector once theuser has modified the gene. Such a kit may also include a DNA ligasethat can be used to catalyze the formation of covalent phosphodiesterlinkages between the modified nuclease-encoding polynucleotide and thetarget vector. A kit described herein may also provide a polynucleotideencoding an antagonistic TNFR2 polypeptide, as well as a package insertdescribing the methods one can use to selectively cleave a particularDNA sequence in the genome of the cell in order to incorporate thepolynucleotide encoding an antagonistic TNFR2 antibody into the genomeat this site. Optionally, the kit may provide a polynucleotide encodinga fusion protein that contains an antagonistic TNFR2 antibody orfragment thereof and an additional polypeptide, such as, e.g., thosedescribed herein.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a description of how the compositions and methodsclaimed herein are performed, made, and evaluated, and are intended tobe purely exemplary described herein and are not intended to limit thescope of what the inventor regards as her invention.

Example 1. Mapping the Discrete Epitopes within TNFR2 that are Bound byAntagonistic TNFR2 Polypeptides

Libraries of linear, cyclic, and bicyclic peptides derived from humanTNFR2 were screened for distinct sequences within the protein thatexhibit high affinity for TNFR2 antibody TNFRAB4. In order to screenconformational epitopes within TFNR2, peptides from distinct regions ofthe primary protein sequence were conjugated to one another to formchimeric peptides. These peptides contained cysteine residues atstrategic positions within their primary sequences. This facilitated anintramolecular cross-linking strategy that was used to constrainindividual peptides to a one of a wide array of three-dimensionalconformations. Unprotected thiols of cysteine residues were cross-linkedvia nucleophilic substitution reactions with divalent and trivalentelectrophiles, such as 2,6-bis(bromomethyl)pyridine and1,3,5-tris(bromomethyl)benzene, so as to form conformationallyrestricted cyclic and bicyclic peptides, respectively. In this way,peptides containing unique combinations of amino acids from disparateregions of the TNFR2 primary sequence were constrained so as tostructurally pre-organize epitopes that may resemble those presented inthe native TNFR2 tertiary structure. Libraries containing these peptideswere screened by immobilizing peptides to distinct regions of a solidsurface and treating the surface in turn with an antagonistic TNFR2antibody, such as TNFRAB1, TNFRAB2, TNFRAB3, TNFRAB4, or TNFRAB5,followed by a secondary antibody conjugated to horseradish peroxidase(HRP), and HRP substrate (2,2′-azino-di-3-ethylbenzthiazoline sulfonate)in the presence of hydrogen peroxide. The solid surface was washed inbetween treatment with successive reagents so as to remove excess ornon-specifically bound materials. The luminescence of each region ofeach surface was subsequently analyzed using a charge coupled device(CCD)-camera and an image processing system.

The “Constrained Libraries of Peptides on Surfaces” (CLIPS) platformstarts with the conversion of the target protein, e.g., TNFR2, into alibrary of up to 10,000 overlapping peptide constructs, using acombinatorial matrix design (Timmerman et al., J. Mol. Recognit., 20:283-29, 2007). On a solid carrier, a matrix of linear peptides issynthesized, which are subsequently shaped into spatially defined CLIPSconstructs. Constructs representing multiple parts of the discontinuousepitope in the correct conformation bind the antibody with highaffinity, which is detected and quantified. Constructs presenting theincomplete epitope bind the antibody with lower affinity, whereasconstructs not containing the epitope do not bind at all. Affinityinformation is used in iterative screens to define the sequence andconformation of epitopes in detail. The raw luminescence data obtainedfrom these ELISA experiments informed the analysis of epitopes presenton the surface of TNFR2 that bind antagonistic TNFR2 antibodies.

Peptide Synthesis

To reconstruct epitopes of the target molecule a library of peptides wassynthesized. An amino functionalized polypropylene support was obtainedby grafting a proprietary hydrophilic polymer formulation via reactionwith t-butyloxycarbonyl-hexamethylenediamine (BocHMDA) usingdicyclohexylcarbodiimide (DCC) with N-hydroxybenzotriazole (HOBt) andsubsequent cleavage of the Boc-groups using trifluoroacetic acid (TFA).Standard Fmoc-peptide synthesis was used to synthesize peptides on theamino-functionalized solid support by custom modified JANUS® liquidhandling stations (Perkin Elmer). CLIPS technology allows one tostructure peptides into single loops, double-loops, triple loops,sheet-like folds, helix-like folds and combinations thereof. CLIPStemplates are coupled to cysteine residues. The side-chains of multiplecysteines in the peptides are coupled to one or two CLIPS templates. Forexample, a 0.5 mM solution of the CLIPS template(2,6-bis(bromomethyl)pyridine) is dissolved in ammonium bicarbonate (20mM, pH 7.8)/acetonitrile (1:3(v/v)). This solution is added to asurface-bound peptide array. The CLIPS template will react withside-chains of two cysteines as present in the solid-phase boundpeptides of the peptide-arrays (455 wells plate with 3 μl wells). Thepeptide arrays are gently shaken in the solution for 30 to 60 minuteswhile completely covered in solution. Finally, the peptide arrays arewashed extensively with excess of H₂O and sonicated in disrupt-buffercontaining 1% SDS/0.1% beta-mercaptoethanol in PBS (pH 7.2) at 70° C.for 30 minutes, followed by sonication in H₂O for another 45 minutes.

Analysis of Binding Affinities of Antagonistic TNFR2 Antibodies bySurface Plasmon Resonance

The affinities of antagonistic TNFR2 antibodies for recombinant humanTNFR2 were measured using BIACORE™ Analysis Services (PrecisionAntibody). Briefly, the antibody was biotinylated at a 5:1stoichiometric ratio using biotinyl-LC-LC-NOSE (Thermo-Fisher) in PBS.Excess biotinylation reagent was removed by centrifugationchromatography and the biotinylated antibody was captured on 3000 RU ofstreptavidin surface to a level of 100 RU. Theoretical maximum of signalwith TNFR2 with that level of antibody capture was 26 RU and that signalwas reached with a preliminary experiment using 500 nM TNFR2 in therunning buffer. Analysis of the kinetics of antigen binding wasperformed at a flow of 60 μL/min with 2 min injections. Antibodies wereinjected at a concentration of 1 mg/ml to the final capture of 100 RU.The instrument used was BIACORE™ 3000 with the BioCap chip (GEHealthcare). Double reference method was used for analysis. Referencechannel contained the identical level of streptavidin.

ELISA Screening

The binding of antibody to each of the synthesized peptides was testedin an ELISA format. Surface-immobilized peptide arrays were incubatedwith primary antibody solution (overnight at 4° C.). After washing, thepeptide arrays were incubated with a 1/1000 dilution of an appropriateantibody peroxidase conjugate (SBA) for one hour at 25° C. Afterwashing, the peroxidase substrate 2,2′-azino-di-3-ethylbenzthiazolinesulfonate (ABTS) and 2 μl/ml of 3 percent H₂O₂ were added. After onehour, the color development was measured. The color development wasquantified with a charge coupled device (CCD)-camera and an imageprocessing system. The values obtained from the CCD camera range from 0to 3000 mAU, similar to a standard 96-well plate ELISA-reader. Theresults are quantified and stored into the Peplab database. Occasionallya well contains an air-bubble resulting in a false-positive value, thecards are manually inspected and any values caused by an air-bubble arescored as 0.

To verify the quality of the synthesized peptides, a separate set ofpositive and negative control peptides was synthesized in parallel.These were screened with a negative control, antibody 57.9, which doesnot specifically bind TNFR2 (Posthumus et al. (J. Virology.64:3304-3309, 1990)).

Epitope Mapping

ELISA was also used to determine linear epitopes present on theextracellular surface of TNFR2. Linear peptides corresponding to variousregions within the TNFR2 primary sequence were purchased from GenScript(Piscataway, N.J.), diluted in coating buffer and placed on Immulon 4HBXFlat Bottom Microtiter Plates (Thermo Scientific) at a concentration of1 μg/well. Primary TNFR2 antagonistic antibodies (0.1 μg/well) wereincubated with substrates. Secondary antibodies against rodent IgG wereused to detect the primary antibodies. Absorbance was measured using theSPECTRAMAX® 190 Absorbance Plate Reader and analyzed with SoftMax Pro6.3 (Molecular Devices).

Results of the epitope mapping analysis are shown in FIG. 1 , whichdisplays the primary structure of human TNFR2 highlighting the regionsthat are bound by exemplary antagonistic TNFR2 antibodies TNFRAB1,TNFRAB2, TNFRAB3, TNFRAB4, and TNFRAB5.

Example 2. Antagonistic TNFR2 Polypeptides of the Human IgG2-A IsoformOptimally Kill T-Reg Cells, Expand T Effector Cells, and Deplete TNFR2+Cancer Cells

Chimeric variants of monoclonal antibody TNFRAB2 containing the variabledomain of TNFRAB2 and the constant domain of various human IgG antibodyisotypes were manufactured. TNFRAB2 is a murine antibody having an IgG2isotype, and is a dominant TNFR2 antagonist. The TNFR2-bindingproperties of TNFRAB2 are described above. TNFRAB2 contains thefollowing CDRs:

TNFRAB2 CDR-H1: (SEQ ID NO: 274) GYTFTDYL TNFRAB2 CDR-H2:(SEQ ID NO: 258) VDPEYGST TNFRAB2 CDR-H3: (SEQ ID NO: 259)ARDDGSYSPFDYWG TNFRAB2 CDR-L1: (SEQ ID NO: 260) QNINKY TNFRAB2 CDR-L2:TYS TNFRAB2 CDR-L3: (SEQ ID NO: 272) CLQYVNLLT

The chimeric antibodies were generated using one of three differenthuman IgG constant domain subtypes: IgG1, IgG2, and IgG3. To investigatethe effects of the chimeric antibodies, nucleic acids encoding theseantibodies were generated using molecular biology techniques, such asthose described herein, and the encoded antibodies were subsequentlyexpressed from host cells prior to evaluation in vitro.

A series of experiments were conducted to assess the TNFR2 antagonisticproperties of these chimeric antibodies, in particular, the ability tokill T-reg cells, induce T effector cell expansion, and to kill TNFR2+SW480 colon cancer cells. FIGS. 2-4 demonstrate these properties forchimeric antibodies having a human IgG1 subtype, and FIGS. 5-7demonstrate these properties for chimeric antibodies having a human IgG2subtype. The effects of chimeric antibodies having a human IgG3 subtypeare reported in Table 3, below, which also provides a summary of thecharacteristics observed for the IgG1 and IgG2 subtypes.

TABLE 3 Effects of chimeric TNFR2 antagonist antibodies on T-reg cellsand effector T cells Antibody isotype and spacing between Effector Tcell antigen-binding T-reg cell killing proliferation arms observed?observed? Human IgG1 (117 Å)  No (0/5)  No (0/5) Human IgG2 (134 Å) Yes(4/5) Yes (3/5) Human IgG3 (125 Å) Little (3/5)   Little (2/5)  In Table 3, T-reg killing and T effector expansion properties arereported qualitatively. Values in parentheses indicate the number ofinstances a particular effect (e.g., T-reg cell killing or T effectorexpansion) was observed out of n=5 individual experiments.

As shown in FIGS. 2-7 and in Table 3, chimeric antibodies having a humanIgG2 isotype were found to exhibit a superior ability to kill T-regcells and TNFR2+ cancer cells and to expand T effector cells relative tochimeric antibodies having either a human IgG1 isotype constant domainor an IgG3 isotype constant domain. Table 3, above, correlates antibodyisotype with the spacing between antigen-binding arms of the respectiveantibody. Collectively, these data demonstrate that a minimum spacingbetween antigen-binding arms is required for an optimal TNFR2 antagonistphenotype. For example, based on the results of these experiments,antibodies having optimal TNFR2 antagonist properties can be developedby matching the distance between antigen-binding arms to the distancebetween antigen-binding arms present in human IgG2 antibodies.

Despite the superior results of chimeric antibodies having the humanIgG2 isotype constant domain, relative to those having the human IgG1 orIgG3 isotype constant domain, it was observed that chimeric antibodieshaving the human IgG2 subtype exhibited a bimodal effect on T-reg cells,T effector cells, and TNFR2+ cancer cells. Namely, as the concentrationof the IgG2 antibody increased in these assays, the value being measuredappears to vary in one direction and then change course. For example, inFIG. 5 , the quantity of T effector cells appears to decrease at a lowerconcentration of the antibody and then to increase as the concentrationof the antibody increases. Similarly, in FIGS. 6 and 7 , the quantity ofT-reg cells and TNFR2+ cancer cells, respectively, appears to increasewith lower concentrations of the antibody and then to decrease withelevated concentrations of the antibody. This behavior inspired aninvestigation into the structure of the chimeric IgG2 antibody.

Upon conducting a polyacrylamide gel electrophoresis separation of thechimeric IgG2 antibody under non-reducing conditions, four unique bandswere observed. These results are shown in FIG. 12 . These four bandscorrespond to four distinct disulfide-bonded isoforms of the human IgG2isotype, which are illustrated in FIGS. 13A-13D. To assess the TNFR2antagonist properties of these disulfide-bonded isoforms, the individualbands were purified and subjected to the T-reg killing, T effectorexpansion, and TNFR2+ cancer cell killing assays described above. Theresults of these experiments are shown in Table 4.

TABLE 4 Effects of human IgG2 disulfide-bonded isoform on the ability ofTNFR2 antagonist antibodies to kill T-reg cells and expand effector Tcells Human IgG2 Effector T cell Disulfide-bonded T-reg cell killingproliferation isoform observed? observed? Band 1 Yes (5/5) Yes (5/5)Band 2 Yes (3/5) Yes (3/5) Band 3  No (1/5)  No (2/5) Band 4  No (0/5) No (0/5)Surprisingly, as shown in Table 4, the disulfide-bonded isoform presentin band 1 of the gel shown in FIG. 12 exhibited superior TNFR2antagonist effects relative to the disulfide-bonded isoforms present inbands 3 and 4. The disulfide-bonded isoform present in band 2 exhibitedgreater antagonistic activity relative to bands 3 and 4 as well. It wasdetermined that band 1 corresponded to the IgG2-A disulfide-bondedisoform and that band 2 corresponded to the IgG2-B disulfide-bondedisoform. The structures of these isoforms are shown in FIGS. 13A and13B, respectively.

In order to stabilize the IgG2-A isoform, a series of mutations wereintroduced into the human IgG2 hinge region of the chimeric IgG2antibodies based on TNFRAB2. These included the C232S and C233S aminoacid substitutions, which preserve the steric and electronegativityproperties of cysteine at positions 232 and 233 while prohibiting theformation of disulfide bonds that are not present in the IgG2-A isoform.The effects of these mutations on the TNFR2 antagonist properties of thechimeric IgG2 antibodies are shown in Table 5, below.

TABLE 5 Effects of IgG2 hinge region amino acid substitutions on theability of TNFR2 antagonist antibodies to kill T-reg cells and expandeffector T cells Mutations introduced into Effector T cell human IgG2hinge T-reg cell killing proliferation region observed? observed? None(wild-type) Yes (4/5) Yes (3/5) C232S and C233S Yes (5/5) Yes (5/5)C232S Yes (5/5) Yes (4/5) C233S Yes (4/5) Yes (4/5)

As shown in Table 5, the C232S and C233S mutations in the hinge regionof human IgG2 result in antagonist TNFR2 antibodies that exhibitsuperior abilities to kill T-reg cells and to induce T effector cellexpansion. As shown in FIGS. 8-10 , introduction of the C232S and C233Smutations into the human IgG2 hinge region of chimeric antibodies basedon TNFRAB2 no longer exhibit a bimodal effect in T-reg cell killing, Teffector cell expansion, and TNFR2+ cancer cell killing assays. Theability of these mutations to impart IgG2 antibodies with optimal cancercell killing properties is further evidenced by the data shown in FIG.11 , which demonstrates that chimeric antibodies containing a human IgG2isotype featuring C232S and C233S amino acid substitutions exhibit atime-dependent ability to reduce tumor volume.

Taken together, these data demonstrate that TNFR2 antagonist antibodieshaving a human IgG2 isotype, and particularly the IgG2-Adisulfide-bonded isoform, and to a lesser extent, the IgG2-Bdisulfide-bonded isoform, exhibit superior TNFR2 antagonist properties.Using the compositions and methods described herein, a variety oftechniques can be employed to generate antibodies that adopt, inparticular, the IgG2-A disulfide-bonding pattern.

Materials and Methods for T-Reg Killing Assay

-   -   Human T-reg Flow™ Kit (BioLegend, Cat. No. 320401)        -   Cocktail Anti-human CD4 PE-Cy5/CD25 PE (BioLegend, Part No.            78930)        -   Alexa Fluor® 488 Anti-human FOXP3, Clone 259D (BioLegend,            Part No. 79467)        -   Alexa Fluor® 488 Mouse IgG1, k Isotype Ctrl (ICFC), Clone            MOPC-21 (BioLegend, Part No. 79486)        -   FOXP3 Fix/Perm Buffer (4×) (BioLegend, Cat. No. 421401)        -   FOXP3 Perm Buffer (10×) (BioLegend, Cat. No. 421402)    -   PE anti-human CD25, Clone: BC96 (BioLegend, Cat. No. 302606)    -   Alexa Fluor® 488 Anti-human FOXP3, Clone 259D (BioLegend, Cat.        No. 320212)    -   PBS pH 7.4 (1×) (Gibco Cat. No. 10010-023)    -   HBSS (1×) (Gibco Cat. No. 14175-095)    -   FBS (heat inactivated)    -   15 ml tubes    -   Bench top centrifuge with swing bucket rotor for 15 ml tubes        (set speed 1100 rpm or 200 g)

Cultured T-reg cells were treated with varying concentrations of TNFR2antagonist antibodies for set periods of time. Following the incubationof T-reg cells under the conditions described above, the cell countswere determined using flow cytometry analysis. T-reg cells at a densityof 0.2-1×10⁶ cells/100 μl were distributed into a 15-ml conical tube andcentrifuged for 5 minutes in order to pellet the cells. The supernatantwas discarded and cells were resuspended in 100 μl of wash buffer(1×HBSS containing 2% FBS). 5 μl of PE anti-human CD25fluorophore-antibody conjugate were added to this mixture, and the cellswere subsequently vortexed and incubated in the dark for 25 minutes. Thecells were then washed by adding 1 ml of wash buffer and subsequentlycentrifuging for 5 minutes. The supernatant was then discarded and 1 mlof FoxP3 fixation/permeabilization buffer (1:4 dilution of 4×FOXP3Fix/Perm buffer in PBS) was added to the cells. The cells were thenvortexed and incubated in the dark for 20 minutes. Cells weresubsequently centrifuged for 5 minutes and supernatant was discarded.Cells were then resuspended in 1 ml of fresh wash buffer, vortexed, andcentrifuged for 5 minutes. Cells were subsequently resuspended in 1 mlof 1×FOXP3 Perm Buffer (1:10 dilution of 10×FOXP3 Perm Buffer in PBS),vortexed, and incubated in the dark for 15 minutes. Followingincubation, cells were centrifuged for 5 minutes and supernatant wassubsequently discarded. The cell pellet was then resuspended in 100 μlof 1×FOXP3 Perm Buffer. At this point, 5 μl of either Alexa Fluor® 488anti-human FOXP3 or Alexa Fluor® 488 mouse IgG1, k isotype control wereadded to the cells. Cells were then vortexed and incubated in the darkfor 35 minutes. Following incubation, cells were washed by adding 1 mlof fresh wash buffer to the cells, vortexing the cells and centrifugingfor 5 minutes. The supernatant was then discarded and the cell pelletwas resuspended in 0.2-0.5 ml of 1×HBSS free of FBS. Cell counts werethen determined by flow cytometry analysis.

Materials and Methods for T Effector Induction Assay

T effector induction assays described in this example were performed asoutlined in Torrey et al. (Science Signaling 10:462, 2017, thedisclosure of which is incorporated herein by reference in itsentirety).

Materials and Methods for Cancer Cell Killing Assay

SW480 colon cancer cells were cultured in 96-well flat-bottom plates ata concentration of 0.1×10⁶ cells per well in 200 ml of media. Cells weretreated directly with TNFR2 antagonistic antibodies and incubated for upto 21 days with half of the medium renewed every 2 to 3 days.

After incubation, cells were detached from the plate with 0.25%Trypsin-EDTA (Gibco), collected, and stained for FACS analysis or withtrypan blue (Sigma-Aldrich) to count viable cells or with cell viabilityassays.

Example 3. Generating Antagonistic TNFR2 Antibodies by Phage Display

An exemplary method for in vitro protein evolution of antagonistic TNFR2antibodies described herein is phage display, a technique which is wellknown in the art. Phage display libraries can be created by making adesigned series of mutations or variations within a coding sequence forthe CDRs of an antibody or the analogous regions of an antibody-likescaffold (e.g., the BC, CD, and DE loops of ¹⁰Fn3 domains). The templateantibody-encoding sequence into which these mutations can be introducedmay be, e.g., a naive human germline sequence as described herein. Thesemutations can be performed using standard mutagenesis techniquesdescribed herein or known in the art. Each mutant sequence thus encodesan antibody corresponding in overall structure to the template excepthaving one or more amino acid variations in the sequence of thetemplate. Retroviral and phage display vectors can be engineered usingstandard vector construction techniques as described herein or known inthe art. P3 phage display vectors along with compatible proteinexpression vectors, as is well known in the art, can be used to generatephage display vectors for antibody diversification as described herein.

The mutated DNA provides sequence diversity, and each transformant phagedisplays one variant of the initial template amino acid sequence encodedby the DNA, leading to a phage population (library) displaying a vastnumber of different but structurally related amino acid sequences. Dueto the well-defined structure of antibody hypervariable regions, theamino acid variations introduced in a phage display screen are expectedto alter the binding properties of the binding peptide or domain withoutsignificantly altering its structure.

In a typical screen, a phage library is contacted with and allowed tobind a TNFR2-derived peptide (e.g., a peptide having the sequence of anyone of SEQ ID NOs: 11, 19, 20, and 34-117), or a particular subcomponentthereof. To facilitate separation of binders and non-binders, it isconvenient to immobilize the target on a solid support. Phage bearing aTNFR2-binding moiety can form a complex with the target on the solidsupport whereas non-binding phage remain in solution and can be washedaway with excess buffer. Bound phage can then liberated from the targetby changing the buffer to an extreme pH (pH 2 or pH 10), changing theionic strength of the buffer, adding denaturants, or other known means.To isolate the binding phage, a protein elution can be performed.

The recovered phage can then be amplified through infection of bacterialcells and the screening process can be repeated with the new pool thatis now depleted in non-binding antibodies and enriched for antibodiesthat bind the target peptide. The recovery of even a few binding phageis sufficient to amplify the phage for a subsequent iteration ofscreening. After a few rounds of selection, the gene sequences encodingthe antibodies or antigen-binding fragments thereof derived fromselected phage clones in the binding pool are determined by conventionalmethods, thus revealing the peptide sequence that imparts bindingaffinity of the phage to the target. During the panning process, thesequence diversity of the population diminishes with each round ofselection until desirable peptide-binding antibodies remain. Thesequences may converge on a small number of related antibodies orantigen-binding fragments thereof, typically 10-50 out of about 10⁹ to10¹⁰ original candidates from each library. An increase in the number ofphage recovered at each round of selection is a good indication thatconvergence of the library has occurred in a screen. After a set ofbinding polypeptides is identified, the sequence information can be usedto design other secondary phage libraries, biased for members havingadditional desired properties (see, e.g., WO 2014/152660; the disclosureof which is incorporated herein by reference).

Example 4. Producing a Humanized Antagonistic TNFR2 Antibody

One method for producing humanized TNFR2 antibodies described herein isto import one or more, or all, of the CDRs of a non-human antagonisticTNFR2 antibody, such as TNFRAB1, TNFRAB2, TNFRAB3, TNFRAB4, or TNFRAB5,into a human antibody consensus sequence. Consensus human antibody heavychain and light chain sequences are known in the art (see, e.g., the“VBASE” human germline sequence database; Kabat et al. (Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242, 1991); Tomlinsonet al. (J. Mol. Biol. 227:776-798, 1992); and Cox et al. (Eur. J.Immunol. 24:827-836, 1994); the disclosures of each of which areincorporated herein by reference). Using established procedures, one canidentify the variable domain framework residues and CDRs of a consensusantibody sequence (e.g., by sequence alignment (see Kabat, supra)). Onecan substitute, e.g., one or more, or all, of the CDR-H1, CDR-H2,CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences of the consensus antibodywith the corresponding CDR sequence(s) of a non-human antagonistic TNFR2antibody described herein in order to produce a humanized, antagonisticTNFR2 antibody described herein. Polynucleotides encoding theabove-described CDRs sequences can be produced synthetically orrecombinantly, e.g., using the techniques described herein or known inthe art.

One example of a variable domain of a consensus human antibody includesthe heavy chain variable domainEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAPG KG LEWVAVISENGSDTYYADSVKG RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARDRGGAVSYFDVWGQGTLVTVSS (SEQ ID NO: 32)and the light chain variable domainDIQMTQSPSSLSASVGDRVTITCRASQDVSSYLAWYQQKPGKAPKLLIYAASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSLPYTFGQGTKVEIKRT (SEQ ID NO: 33), identified inU.S. Pat. No. 6,054,297; the disclosure of which is incorporated hereinby reference (CDRs are shown in bold). In order to produce a humanized,antagonistic TNFR2 antibody of the present disclosure, one canrecombinantly express a polynucleotide encoding the variable domains ofthe above consensus sequences in which one or more, or all, of theCDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences arereplaced with the corresponding CDR sequences of a non-humanantagonistic TNFR2 antibody described herein, such as TNFRAB1, TNFRAB2,TNFRAB3, TNFRAB4, or TNFRAB5, such as a CDR-H1 having the amino acidsequence of any one of SEQ ID NOs: 23, 257, 274, 275, 293, 294, or 295.

A polynucleotide encoding the above heavy chain and light chain variabledomains operatively linked to one another can be incorporated into anexpression vector (e.g., an expression vector optimized for proteinexpression in prokaryotic or eukaryotic cells as described herein orknown in the art). For example, the polynucleotide may contain a regionthat encodes the CDR-H1 of TNFRAB1, TNFRAB2, TNFRAB3, TNFRAB4, orTNFRAB5, such as a CDR-H1 having the amino acid sequence of any one ofSEQ ID NOs: 23, 257, 274, 275, 293, 294, or 295. The humanized antibodycan be expressed in a host cell and subsequently purified from the hostcell medium or the host cell using established techniques, such assize-exclusion chromatography and/or affinity chromatography asdescribed herein.

Example 5. Humanized TNFR2 Antibodies Containing C232S and C233S AminoAcid Substitutions

Using humanization techniques described herein, a series of 25 humanizedTNFR2 antibodies were developed by humanization of monoclonal murineantibody TNFRAB2. These humanized TNFR2 antibodies have an IgG2 isotypeand contain C232S and C233S amino acid substitutions within the IgG2hinge region. As described herein, these substitutions confer beneficialproperties to TNFR2 antibodies, including a heightened ability to killTreg cells and proliferate CD8+ effector T cells. To investigate theeffects of C232S and C233S amino acid substitutions on humanized,antagonistic TNFR2 antibodies having an IgG2 subtype, all 25 humanizedantibodies were tested for antagonistic activity in at least threefunctional assays. First, all humanized antibodies were tested for theirability to kill Treg cells obtained from human blood donors in adose-dependent manner. All humanized antibodies were then tested fortheir ability to proliferate T effector cells from human blood donors.The humanized antibodies were additionally tested for their ability tokill at least one TNFR2-expressing tumor cell line, such as the SW480 orMOTN-1 cell line.

All of the 25 humanized TNFR2 antibodies containing the C232S and C233Ssubstitutions within the IgG2 hinge region exhibited the ability to killTreg cells, expand CD8+ effector T cells, and kill TNFR2-expressingcancer cells. These results are summarized in Table 6, below.

TABLE 6Ability of Humanized TNFR2 Antibodies Containing C232S and C233S Substitutionsin the IgG2 Hinge Region to Deplete Treg Cells, Expand CD8+ T Effector Cells,and Kill TNFR2-expressing Cancer Cells Kills Induces SW480 Kills CD8+ Tor Antibody Light Chain Amino Heavy Chain Amino Treg Cell MOTN-1 No.Acid Sequence Acid Sequence Cells? Growth? Cells?  1 MVSSAQFLGLLLLCFQMGWTLVFLFLLSVTAGVH Yes Yes Yes; GTRCDIQMTQSPSSLS SQVQLVQSGAEVKKPGA SW480ASVGDRVTVTCQASQ SVKVSCKASGYTFTDYLM and NINKYIAWYQQKPGKA HWVRQAPGQGMOTN-1 PKLLIHYTSTLESGVPS LEWIGWVDPEYGSTDYAE RFSGSGSGTDFTLTISS KFKKLQAEDVATYYCLQYVN WVTVTRDTSISTAYMELS LITFGGGTKVEIKRTVA RLTSDDTAVYYCARDDGSAPSVFIFPPSDEQLKSG YSPFDYWGQGTMVTVSS TASVVCLLNNFYPREA ASTKGPSVFPLAPCSRSTKVQWKVDNALQSGNS SESTAALGCLVKDYFPEP QESVTEQDSKDSTYSL VTVSWNSGALTSGVHTFPSSTLTLSKADYEKHKV AVLQSSGLYSLSSVVTVP YACEVTHQGLSSPVTK SSNFGTQTYTCNVDHKPSSFNRGEC NTKVDKTVERKSSVECPP (SEQ ID NO: 297) CPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEY KCKVSNKGLPAPIE KTISKTKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPMLDSDGSFFL YSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 302)  2 MVSSAQFLGLLLLCFQ MGWTLVFLFLLSVTAGVH Yes YesYes; GTRCDIQMTQSPSSLS SEVQLVQSGAEVKKPGAS SW480 ASVGDRVTVTCQASQVKVSCKASGYTFTDYLMH and NINKYIAWYQQKPGKA WVRQAPGQGLEWMGWV MOTN-1PKLLIHYTSTLESGVPS DPEYGSTDYAEKFK RFSGSGSGTDFTLTISS KRVTMTRDTSTSTFYMELLQAEDVATYYCLQYVN SSLRSDDT LITFGGGTKVEIKRTVA AVYFCARDDGSYSPFDYAPSVFIFPPSDEQLKSG WGQGTLVTVSSASTKGP TASVVCLLNNFYPREA SVFPLAPCSRSTSESTAALKVQWKVDNALQSGNS GCLVKDYFPEPVTVSWNS QESVTEQDSKDSTYSL GALTSGVHTFPAVLQSSGSSTLTLSKADYEKHKV LYSLSSVVTVPSSNFGTQ YACEVTHQGLSSPVTK TYTCNVDHKPSNTKVDKTSFNRGEC VERKSSVECPPCPAPPVA (SEQ ID NO: 297) GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV QFNWYVDGVEVHNAKTK PREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSN KGLPAPIEKTISKTKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPM LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK (SEQ ID NO: 303)  3 MVSSAQFLGLLLLCFQMGWTLVFLFLLSVTAGVH Yes Yes Yes; GTRCDIQMTQSPSSLS SEVQLVESGAEVKKPGASSW480 ASVGDRVTVTCQASQ VKVSCKASGYTFTDYLMH and NINKYIAWYQQKPGKAWVRQAPGQGLEWMGWV MOTN-1 PKLLIHYTSTLESGVPS DPEYGSTDYAEKFKKRFSGSGSGTDFTLTISS RVTMTRDTSISTAYMELN LQAEDVATYYCLQYVN RLTSDDTAVYFCARDDGSLITFGGGTKVEIKRTVA YSPFDYWGQGTLVTVSSA APSVFIFPPSDEQLKSGSTKGPSVFPLAPCSRSTS TASVVCLLNNFYPREA ESTAALGCLVKDYFPEPV KVQWKVDNALQSGNSTVSWNSGALTSGVHTFPA QESVTEQDSKDSTYSL VLQSSGLYSLSSVVTVPS SSTLTLSKADYEKHKVSNFGTQTYTCNVDH YACEVTHQGLSSPVTK KPSNTKVDKTVER SFNRGEC KSSVECPPCPAPPVAGPS(SEQ ID NO: 297) VFLFPP KPKDTLMISRTPEVTCVVV DVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNST FRVVSVLTVVHQDWLNGK EYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPP SREEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLY SKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKS LSLSPGK(SEQ ID NO: 304)  4 MVSSAQFLGLLLLCFQ MGWTLVFLFLLSVTAGVH Yes Yes Yes;GTRCDIQMTQSPSSLS SQVQLVQSGTEVTKPGAS SW480 ASVGDRVTVTCQASQVKVSCKASGYTFTDYLMH and NINKYIAWYQQKPGKA WVRQAPGQGLEWLGWV MOTN-1PKLLIHYTSTLESGVPS DPEYGSTDYAEKFKKRVT RFSGSGSGTDFTLTISSMTRDTSTNTVYMELTSLR LQAEDVATYYCLQYVN SEDTAIYYCARDDGSYSP LITFGGGTKVEIKRTVAFDYWGQGTLVTVSSASTK APSVFIFPPSDEQLKSG GPSVFPLAPCSRSTSEST TASVVCLLNNFYPREAAALGCLVKDYFPEPVTVS KVQWKVDNALQSGNS WNSGALTSGVHTFPAVLQ QESVTEQDSKDSTYSLSSGLYSLSSVVTVPSSNF SSTLTLSKADYEKHKV GTQTYTCNVDHKPSNTKV YACEVTHQGLSSPVTKDKTVERKSSVECPPCPAP SFNRGEC PVAGPSVFLFPPKPKDTL (SEQ ID NO: 297)MISRTPEVTCVVVDVSHE DPEVQFNWYVDGVEVHN AKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKC KVSNKGLPAPIEKTISKTK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKT TPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPG K (SEQ ID NO: 305)    5MVSSAQFLGLLLLCFQ MGWTLVFLFLLSVTAGVH Yes Yes Yes; GTRCDIQMTQSPSSLSSEVQLVQSGAEVKKPGAT SW480 ASVGDRVTVTCQASQ VKISCKVSGYTFTDYLMH andNINKYIAWYQQKPGKA WVQQAPGKGLEWMGWV MOTN-1 PKLLIHYTSTLESGVPSDPEYGSTDYAEKFKKRVTI RFSGSGSGTDFTLTISS TADTSTDTAYMELSSLRSLQAEDVATYYCLQYVN EDTAVYYCARDDGSYSPF LITFGGGTKVEIKRTVA DYWGQGVMVTVSSASTKAPSVFIFPPSDEQLKSG GPSVFPLAPCSRSTSEST TASVVCLLNNFYPREA AALGCLVKDYFPEPVTVSKVQWKVDNALQSGNS WNSGALTSGVHTFPAVLQ QESVTEQDSKDSTYSL SSGLYSLSSVVTVPSSNSSTLTLSKADYEKHKV FGTQTYTCNVDH YACEVTHQGLSSPVTK KPSNTKVDKTVER SFNRGECKSSVECPPCPAPPVAGPS (SEQ ID NO: 297) VFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDG VEVHNAKTKPREEQFNST FRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTI SKTKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPMLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK (SEQ ID NO: 306)  6 MVSSAQFLGLLLLCFQMGWTLVFLFLLSVTAGVH Yes Yes Yes; GTRCDIQMTQSPSSLS SQVQLVQSGAEVKKPGAMOTN-1 ASVGDRVTITCQASQNI SVKVSCKASGYTFTDYLM NKYIAWYQQKPGKAPK HWVRQAPGQGLLLYYTSTLESGVPSRF LEWIGWVDPEYGSTDYAE SGSGSGTDYTLTISSLQ KFKKPEDFATYYCLQYVNLIT WVTVTRDTSISTAYMELS FGGGTKVEIKRTVAAP RLTSDDTAVYYCARDDGSSVFIFPPSDEQLKSGTA YSPFDYWGQGTMVTVSS SVVCLLNNFYPREAKV ASTKGPSVFPLAPCSRSTQWKVDNALQSGNSQE SESTAALGCLVKDYFPEP SVTEQDSKDSTYSLSS VTVSWNSGALTSGVHTFPTLTLSKADYEKHKVYA AVLQSSGLYSLSSVVTVP CEVTHQGLSSPVTKSF SSNFGTQTYTCNVDHKPSNRGEC NTKVDKTVERKSSVECPP (SEQ ID NO: 298) CPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEY KCKVSNKGLPAPIE KTISKTKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPMLDSDGSFFL YSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 302)  7 MVSSAQFLGLLLLCFQ MGWTLVFLFLLSVTAGVH Yes YesYes; GTRCDIQMTQSPSSLS SEVQLVQSGAEVKKPGAS SW480 ASVGDRVTITCQASQNIVKVSCKASGYTFTDYLMH and NKYIAWYQQKPGKAPK WVRQAPGQGLEWMGWV MOTN-1LLLYYTSTLESGVPSRF DPEYGSTDYAEKFK SGSGSGTDYTLTISSLQ KRVTMTRDTSTSTFYMELPEDFATYYCLQYVNLIT SSLRSDDT FGGGTKVEIKRTVAAP AVYFCARDDGSYSPFDYSVFIFPPSDEQLKSGTA WGQGTLVTVSSASTKGP SVVCLLNNFYPREAKV SVFPLAPCSRSTSESTAALQWKVDNALQSGNSQE GCLVKDYFPEPVTVSWNS SVTEQDSKDSTYSLSS GALTSGVHTFPAVLQSSGTLTLSKADYEKHKVYA LYSLSSVVTVPSSNFGTQ CEVTHQGLSSPVTKSF TYTCNVDHKPSNTKVDKTNRGEC VERKSSVECPPCPAPPVA (SEQ ID NO: 298) GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV QFNWYVDGVEVHNAKTK PREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSN KGLPAPIEKTISKTKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPM LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK (SEQ ID NO: 303)  8 MVSSAQFLGLLLLCFQMGWTLVFLFLLSVTAGVH Yes Yes Yes; GTRCDIQMTQSPSSLS SEVQLVESGAEVKKPGASSW480 ASVGDRVTITCQASQNI VKVSCKASGYTFTDYLMH NKYIAWYQQKPGKAPKWVRQAPGQGLEWMGWV LLLYYTSTLESGVPSRF DPEYGSTDYAEKFKK SGSGSGTDYTLTISSLQRVTMTRDTSISTAYMELN PEDFATYYCLQYVNLIT RLTSDDTAVYFCARDDGS FGGGTKVEIKRTVAAPYSPFDYWGQGTLVTVSSA SVFIFPPSDEQLKSGTA STKGPSVFPLAPCSRSTS SVVCLLNNFYPREAKVESTAALGCLVKDYFPEPV QWKVDNALQSGNSQE TVSWNSGALTSGVHTFPA SVTEQDSKDSTYSLSSVLQSSGLYSLSSVVTVPS TLTLSKADYEKHKVYA SNFGTQTYTCNVDH CEVTHQGLSSPVTKSFKPSNTKVDKTVER NRGEC KSSVECPPCPAPPVAGPS (SEQ ID NO: 298) VFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVQFNWYVDG VEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGK EYKCKVSNKGLPAPIEK TISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPEN NYKTTPPMLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO: 304)  9 MVSSAQFLGLLLLCFQMGWTLVFLFLLSVTAGVH Yes Yes Yes; GTRCDIQMTQSPSSLS SQVQLVQSGTEVTKPGASSW480 ASVGDRVTITCQASQNI VKVSCKASGYTFTDYLMH NKYIAWYQQKPGKAPKWVRQAPGQGLEWLGWV LLLYYTSTLESGVPSRF DPEYGSTDYAEKFKKRVT SGSGSGTDYTLTISSLQMTRDTSTNTVYMELTSLR PEDFATYYCLQYVNLIT SEDTAIYYCARDDGSYSP FGGGTKVEIKRTVAAPFDYWGQGTLVTVSSASTK SVFIFPPSDEQLKSGTA GPSVFPLAPCSRSTSEST SVVCLLNNFYPREAKVAALGCLVKDYFPEPVTVS QWKVDNALQSGNSQE WNSGALTSGVHTFPAVLQ SVTEQDSKDSTYSLSSSSGLYSLSSVVTVPSSNF TLTLSKADYEKHKVYA GTQTYTCNVDHKPSNTKV CEVTHQGLSSPVTKSFDKTVERKSSVECPPCPAP NRGEC PVAGPSVFLFPPKPKDTL (SEQ ID NO: 298)MISRTPEVTCVVVDVSHE DPEVQFNWYVDGVEVHN AKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKC KVSNKGLPAPIEKTISKTK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKT TPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPG K (SEQ ID NO: 305) 10MVSSAQFLGLLLLCFQ MGWTLVFLFLLSVTAGVH Yes Yes Yes; GTRCDIQMTQSPSSLSSEVQLVQSGAEVKKPGAT SW480 ASVGDRVTITCQASQNI VKISCKVSGYTFTDYLMH andNKYIAWYQQKPGKAPK WVQQAPGKGLEWMGWV MOTN-1 LLLYYTSTLESGVPSRFDPEYGSTDYAEKFKKRVTI SGSGSGTDYTLTISSLQ TADTSTDTAYMELSSLRSPEDFATYYCLQYVNLIT EDTAVYYCARDDGSYSPF FGGGTKVEIKRTVAAP DYWGQGVMVTVSSASTKSVFIFPPSDEQLKSGTA GPSVFPLAPCSRSTSEST SVVCLLNNFYPREAKV AALGCLVKDYFPEPVTVSQWKVDNALQSGNSQE WNSGALTSGVHTFPAVLQ SVTEQDSKDSTYSLSS SSGLYSLSSVVTVPSSNTLTLSKADYEKHKVYA FGTQTYTCNVDH CEVTHQGLSSPVTKSF KPSNTKVDKTVER NRGECKSSVECPPCPAPPVAGPS (SEQ ID NO: 298) VFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDG VEVHNAKTKPREEQFNST FRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTI SKTKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPMLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK (SEQ ID NO: 306) 11 MVSSAQFLGLLLLCFQMGWTLVFLFLLSVTAGVH Yes Yes Yes; GTRCDIQMTQSPSSLS SQVQLVQSGAEVKKPGA SW480ASVGDRVTITCQASQNI SVKVSCKASGYTFTDYLM NKYIAWYQQKPGKVPT HWVRQAPGQGLLIFYTSTLESGVPSRF LEWIGWVDPEYGSTDYAE SGSGSGTDFTLTISSLQ KFKKSEDVATYFCLQYVNLIT WVTVTRDTSISTAYMELS FGGGTKVEIKRTVAAP RLTSDDTAVYYCARDDGSSVFIFPPSDEQLKSGTA YSPFDYWGQGTMVTVSS SVVCLLNNFYPREAKV ASTKGPSVFPLAPCSRSTQWKVDNALQSGNSQE SESTAALGCLVKDYFPEP SVTEQDSKDSTYSLSS VTVSWNSGALTSGVHTFPTLTLSKADYEKHKVYA AVLQSSGLYSLSSVVTVP CEVTHQGLSSPVTKSF SSNFGTQTYTCNVDHKPSNRGEC NTKVDKTVERKSSVECPP (SEQ ID NO: 299) CPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEY KCKVSNKGLPAPIE KTISKTKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPMLDSDGSFFL YSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 302) 12 MVSSAQFLGLLLLCFQ MGWTLVFLFLLSVTAGVH Yes YesYes; GTRCDIQMTQSPSSLS SEVQLVQSGAEVKKPGAS SW480 ASVGDRVTITCQASQNIVKVSCKASGYTFTDYLMH and NKYIAWYQQKPGKVPT WVRQAPGQGLEWMGWV MOTN-1LLIFYTSTLESGVPSRF DPEYGSTDYAEKFK SGSGSGTDFTLTISSLQ KRVTMTRDTSTSTFYMELSEDVATYFCLQYVNLIT SSLRSDDT FGGGTKVEIKRTVAAP AVYFCARDDGSYSPFDYSVFIFPPSDEQLKSGTA WGQGTLVTVSSASTKGP SVVCLLNNFYPREAKV SVFPLAPCSRSTSESTAALQWKVDNALQSGNSQE GCLVKDYFPEPVTVSWNS SVTEQDSKDSTYSLSS GALTSGVHTFPAVLQSSGTLTLSKADYEKHKVYA LYSLSSVVTVPSSNFGTQ CEVTHQGLSSPVTKSF TYTCNVDHKPSNTKVDKTNRGEC VERKSSVECPPCPAPPVA (SEQ ID NO: 299) GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV QFNWYVDGVEVHNAKTK PREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSN KGLPAPIEKTISKTKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPM LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK (SEQ ID NO: 303) 13 MVSSAQFLGLLLLCFQMGWTLVFLFLLSVTAGVH Yes Yes Yes; GTRCDIQMTQSPSSLS SEVQLVESGAEVKKPGASSW480 ASVGDRVTITCQASQNI VKVSCKASGYTFTDYLMH and NKYIAWYQQKPGKVPTWVRQAPGQGLEWMGWV MOTN-1 LLIFYTSTLESGVPSRF DPEYGSTDYAEKFKKSGSGSGTDFTLTISSLQ RVTMTRDTSISTAYMELN SEDVATYFCLQYVNLITRLTSDDTAVYFCARDDGS FGGGTKVEIKRTVAAP YSPFDYWGQGTLVTVSSA SVFIFPPSDEQLKSGTASTKGPSVFPLAPCSRSTS SVVCLLNNFYPREAKV ESTAALGCLVKDYFPEPV QWKVDNALQSGNSQETVSWNSGALTSGVHTFPA SVTEQDSKDSTYSLSS VLQSSGLYSLSSVVTVPS TLTLSKADYEKHKVYASNFGTQTYTCNVDH CEVTHQGLSSPVTKSF KPSNTKVDKTVER NRGEC KSSVECPPCPAPPVAGPS(SEQ ID NO: 299) VFLFPP KPKDTLMISRTPEVTCVVV DVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNST FRVVSVLTVVHQDWLNGK EYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPP SREEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLY SKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKS LSLSPGK(SEQ ID NO: 304) 14 MVSSAQFLGLLLLCFQ MGWTLVFLFLLSVTAGVH Yes Yes Yes;GTRCDIQMTQSPSSLS SQVQLVQSGTEVTKPGAS SW480 ASVGDRVTITCQASQNIVKVSCKASGYTFTDYLMH NKYIAWYQQKPGKVPT WVRQAPGQGLEWLGWV LLIFYTSTLESGVPSRFDPEYGSTDYAEKFKKRVT SGSGSGTDFTLTISSLQ MTRDTSTNTVYMELTSLRSEDVATYFCLQYVNLIT SEDTAIYYCARDDGSYSP FGGGTKVEIKRTVAAP FDYWGQGTLVTVSSASTKSVFIFPPSDEQLKSGTA GPSVFPLAPCSRSTSEST SVVCLLNNFYPREAKV AALGCLVKDYFPEPVTVSQWKVDNALQSGNSQE WNSGALTSGVHTFPAVLQ SVTEQDSKDSTYSLSS SSGLYSLSSVVTVPSSNFTLTLSKADYEKHKVYA GTQTYTCNVDHKPSNTKV CEVTHQGLSSPVTKSF DKTVERKSSVECPPCPAPNRGEC PVAGPSVFLFPPKPKDTL (SEQ ID NO: 299) MISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHN AKTKPREEQFNSTFRVVS VLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK GQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPMLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K (SEQ ID NO: 305) 15 MVSSAQFLGLLLLCFQMGWTLVFLFLLSVTAGVH Yes Yes Yes; GTRCDIQMTQSPSSLS SEVQLVQSGAEVKKPGATSW480 ASVGDRVTITCQASQNI VKISCKVSGYTFTDYLMH NKYIAWYQQKPGKVPTWVQQAPGKGLEWMGWV LLIFYTSTLESGVPSRF DPEYGSTDYAEKFKKRVTI SGSGSGTDFTLTISSLQTADTSTDTAYMELSSLRS SEDVATYFCLQYVNLIT EDTAVYYCARDDGSYSPF FGGGTKVEIKRTVAAPDYWGQGVMVTVSSASTK SVFIFPPSDEQLKSGTA GPSVFPLAPCSRSTSEST SVVCLLNNFYPREAKVAALGCLVKDYFPEPVTVS QWKVDNALQSGNSQE WNSGALTSGVHTFPAVLQ SVTEQDSKDSTYSLSSSSGLYSLSSVVTVPSSN TLTLSKADYEKHKVYA FGTQTYTCNVDH CEVTHQGLSSPVTKSFKPSNTKVDKTVER NRGEC KSSVECPPCPAPPVAGPS (SEQ ID NO: 299) VFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVQFNWYVDG VEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGK EYKCKVSNKGLPAPIEKTI SKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENN YKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSL SPGK (SEQ ID NO: 306) 16MVSSAQFLGLLLLCFQ MGWTLVFLFLLSVTAGVH Yes Yes Yes; GTRCDIQMTQSPSSLSSQVQLVQSGAEVKKPGA SW480 ASIGDRVTITCQASQNI SVKVSCKASGYTFTDYLM andNKYIAWYQQKPGKAPK HWVRQAPGQG MOTN-1 LLIYYTSTLESGVPSRF LEWIGWVDPEYGSTDYAESGSGSGTDFTFTISSLQ KFKK PEDIGTYYCLQYVNLIT WVTVTRDTSISTAYMELSFGQGTRLEIKRTVAAP RLTSDDTAVYYCARDDGS SVFIFPPSDEQLKSGTA YSPFDYWGQGTMVTVSSSVVCLLNNFYPREAKV ASTKGPSVFPLAPCSRST QWKVDNALQSGNSQE SESTAALGCLVKDYFPEPSVTEQDSKDSTYSLSS VTVSWNSGALTSGVHTFP TLTLSKADYEKHKVYA AVLQSSGLYSLSSVVTVPCEVTHQGLSSPVTKSF SSNFGTQTYTCNVDHKPS NRGEC NTKVDKTVERKSSVECPP(SEQ ID NO: 300) CPAPPVAGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTFR VVSVLTVVHQDWLNGKEY KCKVSNKGLPAPIEKTISKTKGQPREPQVYTLP PSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFL YSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQK SLSLSPGK(SEQ ID NO: 302) 17 MVSSAQFLGLLLLCFQ MGWTLVFLFLLSVTAGVH Yes Yes Yes;GTRCDIQMTQSPSSLS SEVQLVQSGAEVKKPGAS SW480 ASIGDRVTITCQASQNIVKVSCKASGYTFTDYLMH and NKYIAWYQQKPGKAPK WVRQAPGQGLEWMGWV MOTN-1LLIYYTSTLESGVPSRF DPEYGSTDYAEKFK SGSGSGTDFTFTISSLQ KRVTMTRDTSTSTFYMELPEDIGTYYCLQYVNLIT SSLRSDDT FGQGTRLEIKRTVAAP AVYFCARDDGSYSPFDYSVFIFPPSDEQLKSGTA WGQGTLVTVSSASTKGP SVVCLLNNFYPREAKV SVFPLAPCSRSTSESTAALQWKVDNALQSGNSQE GCLVKDYFPEPVTVSWNS SVTEQDSKDSTYSLSS GALTSGVHTFPAVLQSSGTLTLSKADYEKHKVYA LYSLSSVVTVPSSNFGTQ CEVTHQGLSSPVTKSF TYTCNVDHKPSNTKVDKTNRGEC VERKSSVECPPCPAPPVA (SEQ ID NO: 300) GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV QFNWYVDGVEVHNAKTK PREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSN KGLPAPIEKTISKTKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPM LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK (SEQ ID NO: 303) 18 MVSSAQFLGLLLLCFQMGWTLVFLFLLSVTAGVH Yes Yes Yes; GTRCDIQMTQSPSSLS SEVQLVESGAEVKKPGASSW480 ASIGDRVTITCQASQNI VKVSCKASGYTFTDYLMH NKYIAWYQQKPGKAPKWVRQAPGQGLEWMGWV LLIYYTSTLESGVPSRF DPEYGSTDYAEKFKK SGSGSGTDFTFTISSLQRVTMTRDTSISTAYMELN PEDIGTYYCLQYVNLIT RLTSDDTAVYFCARDDGS FGQGTRLEIKRTVAAPYSPFDYWGQGTLVTVSSA SVFIFPPSDEQLKSGTA STKGPSVFPLAPCSRSTS SVVCLLNNFYPREAKVESTAALGCLVKDYFPEPV QWKVDNALQSGNSQE TVSWNSGALTSGVHTFPA SVTEQDSKDSTYSLSSVLQSSGLYSLSSVVTVPS TLTLSKADYEKHKVYA SNFGTQTYTCNVDH CEVTHQGLSSPVTKSFKPSNTKVDKTVER NRGEC KSSVECPPCPAPPVAGPS (SEQ ID NO: 300) VFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVQFNWYVDG VEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGK EYKCKVSNKGLPAPIEK TISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPEN NYKTTPPMLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK (SEQ ID NO: 304) 19 MVSSAQFLGLLLLCFQMGWTLVFLFLLSVTAGVH Yes Yes Yes; GTRCDIQMTQSPSSLS SQVQLVQSGTEVTKPGASSW480 ASIGDRVTITCQASQNI VKVSCKASGYTFTDYLMH and NKYIAWYQQKPGKAPKWVRQAPGQGLEWLGWV MOTN-1 LLIYYTSTLESGVPSRF DPEYGSTDYAEKFKKRVTSGSGSGTDFTFTISSLQ MTRDTSTNTVYMELTSLR PEDIGTYYCLQYVNLITSEDTAIYYCARDDGSYSP FGQGTRLEIKRTVAAP FDYWGQGTLVTVSSASTK SVFIFPPSDEQLKSGTAGPSVFPLAPCSRSTSEST SVVCLLNNFYPREAKV AALGCLVKDYFPEPVTVS QWKVDNALQSGNSQEWNSGALTSGVHTFPAVLQ SVTEQDSKDSTYSLSS SSGLYSLSSVVTVPSSNF TLTLSKADYEKHKVYAGTQTYTCNVDHKPSNTKV CEVTHQGLSSPVTKSF DKTVERKSSVECPPCPAP NRGECPVAGPSVFLFPPKPKDTL (SEQ ID NO: 300) MISRTPEVTCVVVDVSHE DPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVS VLTVVHQDWLNGKEYKC KVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPG K(SEQ ID NO: 305) 20 MVSSAQFLGLLLLCFQ MGWTLVFLFLLSVTAGVH Yes Yes Yes;GTRCDIQMTQSPSSLS SEVQLVQSGAEVKKPGAT SW480 ASIGDRVTITCQASQNIVKISCKVSGYTFTDYLMH NKYIAWYQQKPGKAPK WVQQAPGKGLEWMGWV LLIYYTSTLESGVPSRFDPEYGSTDYAEKFKKRVTI SGSGSGTDFTFTISSLQ TADTSTDTAYMELSSLRSPEDIGTYYCLQYVNLIT EDTAVYYCARDDGSYSPF FGQGTRLEIKRTVAAP DYWGQGVMVTVSSASTKSVFIFPPSDEQLKSGTA GPSVFPLAPCSRSTSEST SVVCLLNNFYPREAKV AALGCLVKDYFPEPVTVSQWKVDNALQSGNSQE WNSGALTSGVHTFPAVLQ SVTEQDSKDSTYSLSS SSGLYSLSSVVTVPSSNTLTLSKADYEKHKVYA FGTQTYTCNVDH CEVTHQGLSSPVTKSF KPSNTKVDKTVER NRGECKSSVECPPCPAPPVAGPS (SEQ ID NO: 300) VFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDG VEVHNAKTKPREEQFNST FRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTI SKTKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPMLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK (SEQ ID NO: 306) 21 MVSSAQFLGLLLLCFQMGWTLVFLFLLSVTAGVH Yes Yes Yes; GTRCDIQMTQSPSSLS SQVQLVQSGAEVKKPGA SW480ASVGDRVTITCQASQNI SVKVSCKASGYTFTDYLM and NKYIAWYQQKPGKAPK HWVRQAPGQGMOTN-1 LLIYYTSTLESGVPSRF LEWIGWVDPEYGSTDYAE SGSGSGTDFTFTISSLQ KFKKPEDIATYYCLQYVNLIT WVTVTRDTSISTAYMELS FGAGTKLELKRTVAAP RLTSDDTAVYYCARDDGSSVFIFPPSDEQLKSGTA YSPFDYWGQGTMVTVSS SVVCLLNNFYPREAKV ASTKGPSVFPLAPCSRSTQWKVDNALQSGNSQE SESTAALGCLVKDYFPEP SVTEQDSKDSTYSLSS VTVSWNSGALTSGVHTFPTLTLSKADYEKHKVYA AVLQSSGLYSLSSVVTVP CEVTHQGLSSPVTKSF SSNFGTQTYTCNVDHKPSNRGEC NTKVDKTVERKSSVECPP (SEQ ID NO: 301) CPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEY KCKVSNKGLPAPIE KTISKTKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPMLDSDGSFFL YSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 302) 22 MVSSAQFLGLLLLCFQ MGWTLVFLFLLSVTAGVH Yes YesYes; GTRCDIQMTQSPSSLS SEVQLVQSGAEVKKPGAS SW480 ASVGDRVTITCQASQNIVKVSCKASGYTFTDYLMH and NKYIAWYQQKPGKAPK WVRQAPGQGLEWMGWV MOTN-1LLIYYTSTLESGVPSRF DPEYGSTDYAEKFK SGSGSGTDFTFTISSLQ KRVTMTRDTSTSTFYMELPEDIATYYCLQYVNLIT SSLRSDDT FGAGTKLELKRTVAAP AVYFCARDDGSYSPFDYSVFIFPPSDEQLKSGTA WGQGTLVTVSSASTKGP SVVCLLNNFYPREAKV SVFPLAPCSRSTSESTAALQWKVDNALQSGNSQE GCLVKDYFPEPVTVSWNS SVTEQDSKDSTYSLSS GALTSGVHTFPAVLQSSGTLTLSKADYEKHKVYA LYSLSSVVTVPSSNFGTQ CEVTHQGLSSPVTKSF TYTCNVDHKPSNTKVDKTNRGEC VERKSSVECPPCPAPPVA (SEQ ID NO: 301) GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV QFNWYVDGVEVHNAKTK PREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSN KGLPAPIEKTISKTKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPM LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK (SEQ ID NO: 303) 23 MVSSAQFLGLLLLCFQMGWTLVFLFLLSVTAGVH Yes Yes Yes; GTRCDIQMTQSPSSLS SEVQLVESGAEVKKPGASSW480 ASVGDRVTITCQASQNI VKVSCKASGYTFTDYLMH and NKYIAWYQQKPGKAPKWVRQAPGQGLEWMGWV MOTN-1 LLIYYTSTLESGVPSRF DPEYGSTDYAEKFKKSGSGSGTDFTFTISSLQ RVTMTRDTSISTAYMELN PEDIATYYCLQYVNLITRLTSDDTAVYFCARDDGS FGAGTKLELKRTVAAP YSPFDYWGQGTLVTVSSA SVFIFPPSDEQLKSGTASTKGPSVFPLAPCSRSTS SVVCLLNNFYPREAKV ESTAALGCLVKDYFPEPV QWKVDNALQSGNSQETVSWNSGALTSGVHTFPA SVTEQDSKDSTYSLSS VLQSSGLYSLSSVVTVPS TLTLSKADYEKHKVYASNFGTQTYTCNVDH CEVTHQGLSSPVTKSF KPSNTKVDKTVER NRGEC KSSVECPPCPAPPVAGPS(SEQ ID NO: 301) VFLFPP KPKDTLMISRTPEVTCVVV DVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNST FRVVSVLTVVHQDWLNGK EYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPP SREEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLY SKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKS LSLSPGK(SEQ ID NO: 304) 24 MVSSAQFLGLLLLCFQ MGWTLVFLFLLSVTAGVH Yes Yes Yes;GTRCDIQMTQSPSSLS SQVQLVQSGTEVTKPGAS SW480 ASVGDRVTITCQASQNIVKVSCKASGYTFTDYLMH and NKYIAWYQQKPGKAPK WVRQAPGQGLEWLGWV MOTN-1LLIYYTSTLESGVPSRF DPEYGSTDYAEKFKKRVT SGSGSGTDFTFTISSLQMTRDTSTNTVYMELTSLR PEDIATYYCLQYVNLIT SEDTAIYYCARDDGSYSP FGAGTKLELKRTVAAPFDYWGQGTLVTVSSASTK SVFIFPPSDEQLKSGTA GPSVFPLAPCSRSTSEST SVVCLLNNFYPREAKVAALGCLVKDYFPEPVTVS QWKVDNALQSGNSQE WNSGALTSGVHTFPAVLQ SVTEQDSKDSTYSLSSSSGLYSLSSVVTVPSSNF TLTLSKADYEKHKVYA GTQTYTCNVDHKPSNTKV CEVTHQGLSSPVTKSFDKTVERKSSVECPPCPAP NRGEC PVAGPSVFLFPPKPKDTL (SEQ ID NO: 301)MISRTPEVTCVVVDVSHE DPEVQFNWYVDGVEVHN AKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKC KVSNKGLPAPIEKTISKTK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKT TPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPG K (SEQ ID NO: 305) 25MVSSAQFLGLLLLCFQ MGWTLVFLFLLSVTAGVH Yes Yes Yes; GTRCDIQMTQSPSSLSSEVQLVQSGAEVKKPGAT SW480 ASVGDRVTITCQASQNI VKISCKVSGYTFTDYLMHNKYIAWYQQKPGKAPK WVQQAPGKGLEWMGWV LLIYYTSTLESGVPSRF DPEYGSTDYAEKFKKRVTISGSGSGTDFTFTISSLQ TADTSTDTAYMELSSLRS PEDIATYYCLQYVNLITEDTAVYYCARDDGSYSPF FGAGTKLELKRTVAAP DYWGQGVMVTVSSASTK SVFIFPPSDEQLKSGTAGPSVFPLAPCSRSTSEST SVVCLLNNFYPREAKV AALGCLVKDYFPEPVTVS QWKVDNALQSGNSQEWNSGALTSGVHTFPAVLQ SVTEQDSKDSTYSLSS SSGLYSLSSVVTVPSSN TLTLSKADYEKHKVYAFGTQTYTCNVDH CEVTHQGLSSPVTKSF KPSNTKVDKTVER NRGEC KSSVECPPCPAPPVAGPS(SEQ ID NO: 301) VFLFPP KPKDTLMISRTPEVTCVVV DVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNST FRVVSVLTVVHQDWLNGK EYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYS KLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSL SPGK(SEQ ID NO: 306)

Example 6. Treatment of Cancer in a Human Patient by Administration ofAntagonistic TNFR2 Polypeptides

The antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides,antibodies, antigen-binding fragments thereof, or constructs thereof)described herein can be administered to a human patient in order totreat a cell proliferation disorder, such as cancer. The antagonisticpolypeptide may contain, for example, a CDR-H1 having the amino acidsequence of any one of SEQ ID NOs: 23, 257, 274, 275, 293, 294, or 295.Administration of these polypeptides may suppress the growth andproliferation of T-reg cells. Thus, antibodies described herein can beadministered to a patient in order to suppress a T-reg-mediated immuneresponse. For instance, a human patient suffering from cancer, e.g., acancer described herein, can be treated by administering an antagonisticTNFR2 polypeptide described herein by an appropriate route (e.g.,intravenously) at a particular dosage (e.g., between 0.001 and 100mg/kg/day) over a course of days, weeks, months, or years. If desired,the antagonistic-TNFR2 polypeptide can be modified, e.g., byhyperglycosylation or by conjugation with PEG, so as to evade immunerecognition and/or to improve the pharmacokinetic profile of thepolypeptide.

The cancer may be, for instance, one that is characterized by cells thatexpress TNFR2, such as, for instance, Hodgkin's lymphoma, cutaneousnon-Hodgkin's lymphoma, T cell lymphoma, ovarian cancer, colon cancer,multiple myeloma, renal cell carcinoma, skin cancer, lung cancer, livercancer, endometrial cancer, a hematopoietic or lymphoid cancer, acentral nervous system cancer (e.g., glioma, neuroblastoma, and othercancers of central nervous system cells described herein), breastcancer, pancreatic cancer, stomach cancer, esophageal cancer, and uppergastrointestinal cancer. In such instances, the antagonistic TNFR2polypeptide may treat the cancer by one or more mechanisms. For example,the antagonistic TNFR2 polypeptide may bind TNFR2 on the surface of aT-reg cell, such as an activated T-reg cell expressing CD25^(Hi) andCD45RA^(Low), or a MDSC, thereby inhibiting the proliferation of, and/ordirectly killing, the T-reg cell or MDSC. The T-reg cells and/or MDSCsthat are killed or for which proliferation is suppressed may be thosethat are located in the microenvironment of a tumor. The reducedpopulation of T-reg cells and/or MDSCs effectuated by the antagonisticTNFR2 polypeptide may, in turn, enable the expansion of populations oftumor-reactive CD8+ cytotoxic T cells, which can mount an immuneresponse against the cancerous cells. The antagonistic TNFR2 polypeptidemay, additionally, or alternatively, induce the direct expansion of CD8+effector T cells. Additionally, or alternatively, the antagonistic TNFR2polypeptide may bind TNFR2 on the surface of a TNFR2+ cancer cell,thereby inhibiting the proliferation of, and/or directly killing, thecancer cell.

The progression of the cancer that is treated with an antagonistic TNFR2polypeptide described herein can be monitored by any one or more ofseveral established methods. A physician can monitor the patient bydirect observation in order to evaluate how the symptoms exhibited bythe patient have changed in response to treatment. A patient may also besubjected to MRI, CT scan, or PET analysis in order to determine if atumor has metastasized or if the size of a tumor has changed, e.g.,decreased in response to treatment with an anti-TNFR2 antibody describedherein. Optionally, cells can be extracted from the patient and aquantitative biochemical analysis can be conducted in order to determinethe relative cell-surface concentrations of various growth factorreceptors, such as the epidermal growth factor receptor. Based on theresults of these analyses, a physician may prescribe higher/lowerdosages or more/less frequent dosing of the antagonistic TNFR2polypeptide in subsequent rounds of treatment.

Example 7. Treatment of HIV in a Human Patient by Administration ofAntagonistic TNFR2 Polypeptides

The antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides,antibodies, antigen-binding fragments thereof, and constructs thereof)described herein can be administered to a human patient in order totreat a viral infection, such as HIV. Administration of thesepolypeptides can, for instance, suppress the growth and proliferation ofT-reg cells and MDSCs, which can enhance the immune response of apatient by allowing the expansion of cytotoxic T lymphocytes capable ofmounting an attack on infected cells. For instance, a human patientsuffering from HIV can be treated by administering an antagonistic TNFR2polypeptide described herein by an appropriate route (e.g.,intravenously) at a particular dosage (e.g., between 0.001 and 100mg/kg/day) over a course of days, weeks, months, or years. Theantagonistic polypeptide may contain, for example, a CDR-H1 having theamino acid sequence of any one of SEQ ID NOs: 23, 257, 274, 275, 293,294, or 295. If desired, the polypeptide can be modified, e.g., byhyperglycosylation or by conjugation with PEG, so as to evade immunerecognition and/or to improve the pharmacokinetic profile of thepolypeptide.

The progression of HIV that is treated with an antagonistic TNFR2polypeptide described herein can be monitored by any one or more ofseveral established methods. A physician can monitor the patient bydirect observation in order to evaluate how the symptoms exhibited bythe patient have changed in response to treatment. A blood sample canalso be withdrawn from the patient in order to analyze the cell count ofone or more white blood cells in order to determine if the quantity ofinfected cells has changed (e.g., decreased) in response to treatmentwith an antagonistic TNFR2 polypeptide described herein. Based on theresults of these analyses, a physician may prescribe higher/lowerdosages or more/less frequent dosing of the antagonistic TNFR2polypeptide in subsequent rounds of treatment.

Example 8. Treatment of Mycobacterium tuberculosis in a Non-Human Mammalby Administration of Antagonistic TNFR2 Polypeptides

The antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides,antibodies, antigen-binding fragments thereof, and constructs thereof)described herein can be administered to a non-human mammal (e.g., abovine mammal, pig, bison, horse, sheep, goat, cow, cat, dog, rabbit,hamster, guinea pig, or other non-human mammal) in order to treat abacterial infection, such as Mycobacterium tuberculosis. Administrationof these polypeptides may, for instance, suppress the proliferation of,and/or directly kill, T-reg cells and/or MDSCs, which can enhance theimmune response of a patient by allowing the expansion of cytotoxic Tlymphocytes capable of mounting an attack on the pathogenic organism.For instance, a non-human mammal suffering from Mycobacteriumtuberculosis can be treated by administering an antagonistic TNFR2polypeptide described herein by an appropriate route (e.g.,intravenously) at a particular dosage (e.g., between 0.001 and 100mg/kg/day) over a course of days, weeks, months, or years. Theantagonistic polypeptide may contain, for example, a CDR-H1 having theamino acid sequence of any one of SEQ ID NOs: 23, 257, 274, 275, 293,294, or 295. If desired, the antagonistic TNFR2 polypeptide can bemodified, e.g., by hyperglycosylation or by conjugation with PEG, so asto evade immune recognition and/or to improve the pharmacokineticprofile of the polypeptide.

The progression of the Mycobacterium tuberculosis infection that istreated with an antagonistic TNFR2 polypeptide described herein can bemonitored by any one or more of several established methods. A physiciancan monitor the patient by direct observation in order to evaluate howthe symptoms exhibited by the patient have changed in response totreatment. A blood sample can also be withdrawn from the patient inorder to analyze the cell count of one or more white blood cells inorder to determine if the immune response has changed (e.g., increased)in response to treatment with an antagonistic TNFR2 polypeptidedescribed herein. Based on the results of these analyses, a physicianmay prescribe higher/lower dosages or more/less frequent dosing of theantagonistic TNFR2 polypeptide in subsequent rounds of treatment.

Example 9. Treatment of Cancer or an Infectious Disease in a HumanPatient by Administration of Antagonistic TNFR2 Polypeptides inCombination with an Immunotherapy Agent

The antagonistic TNFR2 antibodies, antigen-binding fragments,single-chain polypeptides, and constructs described herein can beadministered to a human patient in combination with (for instance,admixed with, co-administered with, or administered separately from) animmunotherapy agent in order to treat a cell proliferation disorder,such as cancer, or an infectious disease, such as a viral, bacterial,fungal, or parasitic infection. Administration of the antibody,antigen-binding fragment, single-chain polypeptide, or construct cansuppress the growth and proliferation of T-reg cells and/or cancer cellsthat express TNFR2. Immunotherapy agents, such as anti-CTLA-4 agents(e.g., an anti-CTLA-4 antibody or antigen-binding fragment thereof, suchas ipilimumab and tremelimumab), anti-PD-1 agents (e.g., an anti-PD-1antibody or antigen-binding fragment thereof, such as nivolumab,pembrolizumab, avelumab, durvalumab, and atezolizumab), anti-PD-L1agents (e.g., atezolizumab and avelumab), anti-PD-L2 agents, TNF-αcross-linking agents, TRAIL cross-linking agents, anti-CD27 agents,anti-CD30 agents, anti-CD40 agents, anti-4-1 BB agent, anti-GITR agents,anti-OX40 agents, anti-TRAILR1 agents, anti-TRAILR2 agent, andanti-TWEAKR agents can function in tandem with antagonist TNFR2antibodies, antigen-binding fragments thereof, single-chainpolypeptides, or constructs, as immunotherapy agents are capable ofdownregulating the signal transduction of immune checkpoint proteins(e.g., immune checkpoint receptors and/or ligands) that would otherwiselead to tolerance toward tumor-associated antigens and downregulation ofthe cytotoxic T cell response. Additional examples of immunotherapyagents that may be used in conjunction with an antagonistic TNFR2antibody, antigen-binding fragment thereof, single-chain polypeptide, orconstruct include Targretin, Interferon-alpha, clobetasol, PegInterferon (e.g., PEGASYS®), prednisone, Romidepsin, Bexarotene,methotrexate, Triamcinolone cream, anti-chemokines, Vorinostat,gabapentin, antibodies to lymphoid cell surface receptors and/orlymphokines, antibodies to surface cancer proteins, and/or smallmolecular therapies like Vorinostat.

A physician of skill in the art may administer a polypeptide describedherein that specifically binds to TNFR2 as an antagonist (e.g., anantibody, antigen-binding fragment thereof, single-chain polypeptide, orconstruct thereof) to a human patient suffering from a cancer orinfectious disease in combination with an immunotherapy agent. Theantagonistic polypeptide may contain, for example, a CDR-H1 having theamino acid sequence of any one of SEQ ID NOs: 23, 257, 274, 275, 293,294, or 295. The polypeptide and the immunotherapy agent may beadministered to the patient by an appropriate route of administration(for example, intravenously, intramuscularly, or subcutaneously, amongothers) at a particular dosage (for example, between 0.001 and 100mg/kg/day, among other ranges) over a course of days, weeks, months, oryears. If desired, the anti-TNFR2 antibody, antigen-binding fragment,single-chain polypeptide, or construct can be modified, for instance, byhyperglycosylation or by conjugation with PEG, so as to evade immunerecognition and/or to improve the pharmacokinetic profile of theantibody, antigen-binding fragment, single-chain polypeptide, orconstruct.

The progression of the cancer or infectious disease that is treated inthis fashion can be monitored by any one or more of several establishedmethods. A physician can monitor the patient by direct observation inorder to evaluate how the symptoms exhibited by the patient have changedin response to treatment. A patient may also be subjected to MRI, CTscan, or PET analysis in order to determine if a tumor has metastasizedor if the size of a tumor has changed, for example, decreased inresponse to treatment with an anti-TNFR2 antibody, antigen-bindingfragment, single-chain polypeptide, or construct and an immunotherapyagent. Optionally, cells can be extracted from the patient and aquantitative biochemical analysis can be conducted in order to determinethe relative cell-surface concentrations of various growth factorreceptors, such as the epidermal growth factor receptor. Based on theresults of these analyses, a physician may prescribe higher/lowerdosages or more/less frequent dosing of the antagonistic TNFR2 antibody,antigen-binding fragment, single-chain polypeptide, or construct andimmunotherapy agent in subsequent rounds of treatment.

Other Embodiments

All publications, patents, and patent applications mentioned in thisspecification are incorporated herein by reference to the same extent asif each independent publication or patent application was specificallyand individually indicated to be incorporated by reference.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations described herein following, in general, theprinciples described herein and including such departures from theinvention that come within known or customary practice within the art towhich the invention pertains and may be applied to the essentialfeatures hereinbefore set forth, and follows in the scope of the claims.

Other embodiments are within the claims.

1. A recombinant anti-tumor necrosis factor receptor (TNFR) superfamilyantibody or antigen-binding fragment thereof, wherein the antibody orantigen-binding fragment thereof comprises: (a) a heavy chain frameworkregion having the amino acid sequence of:MGWTLVFLFLLSVTAGVHSEVQLVQSGAEVKKPGASVKVSCKASXXXXXXXXXXWVRQAPGQGLEWMGWXXXXXXXXXYAEKFKKRVTMTRDTSTSTFYMELSSLRSDDTAVYFCARXXXXXXXXXYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPS;

 and (b) a light chain framework region having the amino acid sequenceof: MVSSAQFLGLLLLCFQGTRCDIQMTQSPSSLSASVGDRVTITCQASXXXXXXIAWYQQKPGKVPTLLIFXXXTLESGVPSRFSGSGSGTDFTLTISSLQSEDVATYFCXXXXXXXXFGGGTKVEIK;

and wherein “X” designates amino acid residues corresponding tocomplementarity-determining regions (CDRs).
 2. The antibody orantigen-binding fragment thereof of claim 1, wherein the antibody orantigen-binding fragment thereof binds to a TNFR superfamily protein. 3.The antibody or antigen-binding fragment thereof of claim 2, wherein theTNFR superfamily protein is TNFR2.
 4. A recombinant anti-TNFRsuperfamily antibody or antigen-binding fragment thereof, wherein theantibody or antigen-binding fragment thereof comprises: (a) a heavychain having the amino acid sequence of:MGWTLVFLFLLSVTAGVHSEVQLVQSGAEVKKPGASVKVSCKASXXXXXXXXXXWVRQAPGQGLEWMGWXXXXXXXXXYAEKFKKRVTMTRDTSTSTFYMELSSLRSDDTAVYFCARXXXXXXXXXYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK;

 and (b) a light chain having the amino acid sequence of:MVSSAQFLGLLLLCFQGTRCDIQMTQSPSSLSASVGDRVTITCQASXXXXXXIAWYQQKPGKVPTLLIFXXXTLESGVPSRFSGSGSGTDFTLTISSLQSEDVATYFCXXXXXXXXFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

and wherein “X” designates amino acid residues corresponding to CDRs. 5.The antibody or antigen-binding fragment thereof of claim 4, wherein theantibody or antigen-binding fragment thereof binds to a TNFR superfamilyprotein.
 6. The antibody or antigen-binding fragment thereof of claim 5,wherein the TNFR superfamily protein is TNFR2.